Novel peptides, combination of peptides as targets and for use in immunotherapy against gallbladder cancer and cholangiocarcinoma and other cancers

ABSTRACT

The present invention relates to peptides, proteins, nucleic acids and cells for use in immunotherapeutic methods. In particular, the present invention relates to the immunotherapy of cancer. The present invention furthermore relates to tumor-associated T-cell peptide epitopes, alone or in combination with other tumor-associated peptides that can for example serve as active pharmaceutical ingredients of vaccine compositions that stimulate anti-tumor immune responses, or to stimulate T cells ex vivo and transfer into patients. Peptides bound to molecules of the major histocompatibility complex (MHC), or peptides as such, can also be targets of antibodies, soluble T-cell receptors, and other binding molecules.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a Continuation of U.S. patent application Ser. No.16/563,740, filed 6 Sep. 2019, which is a Continuation of U.S. patentapplication Ser. No. 15/602,746, filed 23 May 2017, now U.S. Pat. No.10,464,990, issued 5 Nov. 2019, which claims the benefit of U.S.Provisional Application Ser. No. 62/341,367, filed 25 May 2016, andGreat Britain Application No. 1609193.6, filed 25 May 2016, the contentof each of these applications is herein incorporated by reference intheir entirety.

This application also is related to PCT/EP2017/062334 filed 23 May 2017,the content of which is incorporated herein by reference in itsentirety.

REFERENCE TO SEQUENCE LISTING SUBMITTED AS A COMPLIANT ASCII TEXT FILE(.txt)

Pursuant to the EFS-Web legal framework and 37 CFR §§ 1.821-825 (seeMPEP § 2442.03(a)), a Sequence Listing in the form of an ASCII-complianttext file (entitled “Sequence_Listing_2912919-072007_ST25.txt” createdon 5 Mar. 2020, and 6,559 bytes in size) is submitted concurrently withthe instant application, and the entire contents of the Sequence Listingare incorporated herein by reference.

FIELD

The present invention relates to peptides, proteins, nucleic acids andcells for use in immunotherapeutic methods. In particular, the presentinvention relates to the immunotherapy of cancer. The present inventionfurthermore relates to tumor-associated T-cell peptide epitopes, aloneor in combination with other tumor-associated peptides that can forexample serve as active pharmaceutical ingredients of vaccinecompositions that stimulate anti-tumor immune responses, or to stimulateT cells ex vivo and transfer into patients. Peptides bound to moleculesof the major histocompatibility complex (MHC), or peptides as such, canalso be targets of antibodies, soluble T-cell receptors, and otherbinding molecules.

The present invention relates to several novel peptide sequences andtheir variants derived from HLA class I molecules of human tumor cellsthat can be used in vaccine compositions for eliciting anti-tumor immuneresponses, or as targets for the development ofpharmaceutically/immunologically active compounds and cells.

BACKGROUND OF THE INVENTION

The most common form of biliary tract cancer is an adenocarcinoma of thebile duct epithelium and includes cholangiocarcinoma (CCC) andgallbladder adenocarcinoma (GBC). Both diseases are characterized by anincreasing incidence and poor outcome.

Cholangiocarcinoma is the second most common liver cancer afterhepatocellular adenocarcinoma (HCC). Cholangiocarcinoma can develop inany part of the bile duct system and is therefore classified intointrahepatic, perihilar and distal. The incidence varies extremelyworldwide with the highest rates in Northeast Thailand (>80 per 100,000population) and low rates in the Western world (0.3-3 per 100,000)(Bridgewater et al., 2014). Although it is not very common in westerncountries, incidence rates are increasing due to aging populations. InGermany CCC mortality more than tripled between 1998 and 2008 due todemographic changes (von Hahn et al., 2011). The average age of peoplediagnosed with CCC is around 70 years (American Cancer Society, 2015).

Risk factors for cholangiocarcinoma include chronic liver and bile tractdiseases such as primary sclerosing cholangitis, hepatolithiasis, bileduct stones, gallbladder polyps, liver fluke infections, cirrhosis, butalso infections with hepatitis B or C, inflammatory bowel diseases,older age, obesity, exposure to the radioactive substance Thorotrast,family history, diabetes and alcohol consumption (World Cancer Report,2014).

Cholangiocarcinoma is much more common in South-East Asia whereparasitic infections with Clonorchis and Opisthorchis species areendemic. Beyond these regions characterized by a high incidence offoodborne liver flukes causing chronic inflammation of the biliary tree,cholangiocarcinoma is sporadic and still rather uncommon to rare.

Cholangiocarcinoma is mostly identified in advanced stages because it isdifficult to diagnose. Symptoms are unspecific and diagnosis of biliaryorigin remains challenging since there is no specific antigenic marker.Therefore, diagnosis of CCC requires clinical and radiological exclusionof metastasis from other sites. Rising levels of serum markers such asCA19-9 and CEA may be helpful in patients with underlying hepaticdiseases (World Cancer Report, 2014).

Molecular carcinogenesis of CCC includes many known oncogenes andsignaling pathways. Activating KRAS mutations, loss-of-functionmutations of TP53, FGFR2 fusion genes, IDH1/2 mutations,hypermethylation of p16^(INK4A) and SOCS3, JAK-STAT activation,over-expression of EGFR/HER2, aberrant MAPK/ERK activation and c-Metover-expression are commonly found in CCC. The link between chronicbiliary infection and CCC carcinogenesis is thought to be the activationof the IL-6/STAT3 pathway. IL-6 is not only secreted by tumor cellsenhancing cell growth through autocrine mechanisms but also regulatesthe expression of other genes, such as EGFR (World Cancer Report, 2014).However, molecular stratification based on these genetic abnormalitiesis not ready for clinical use (Bridgewater et al., 2014).

Cholangiocarcinoma is difficult to treat and is usually lethal. The onlycurative treatment option is complete resection (R0). Unfortunately,only around 30% of tumors are resectable. Most stage 0, I and II, andsome stage III tumors are resectable depending on their exact location,while other stage III and most stage IV tumors are unresectable(American Cancer Society, 2015). The 5-year survival after curativeresection (R0) is 40%. There is no evidence that adjuvant chemotherapyprolongs 5-year survival after tumor resection. Lymph node involvementis present in one third of patients eligible for surgical treatment andis associated with poor surgical outcome. 5-year survival afternon-curative resection (R1) is 20%. Given its prognostic value,lymphadenectomy of regional lymph nodes is recommended. While N1sometimes still is considered suitable for surgical management, for N2and M1 disease surgery is contraindicated (Bridgewater et al., 2014).

If resection of the tumor is not feasible, treatment options are verylimited. Different palliative chemotherapeutic drugs such as5-fluorouracil, gemcitabine, cisplatin, capecitabine, oxaliplatin are inuse (American Cancer Society, 2015). Standard of care for palliativechemotherapy is combination of gemcitabine and cisplatin. The mediansurvival after chemotherapy is only 12 months.

Liver transplantation can be indicated for patients with early stageunresectable tumors but is discussed controversially.

The efficacy of biological therapies in biliary tract cancers has beenmixed. Drugs targeting blood vessel growth such as Sorafenib,bevacizumab, pazopanib and regorafenib are now studied for the treatmentof CCC. Additionally, drugs that target EGFR such as cetuximab andpanitumumab are used in clinical studies in combination withchemotherapy (American Cancer Society, 2015). For most drugs tested sofar disease control and overall survival were not improved significantlybut there are further clinical trials ongoing.

Gallbladder cancer is the most common and aggressive malignancy of thebiliary tract worldwide. Unspecific clinical presentation also delaysdiagnosis and leads to the fact that only 10% of all patients arecandidates for surgery. Due to the anatomical complexity of the biliarysystem and the high recurrence rate surgery is only curative in theminority of cases. Risk factors are similar to CCC but GBC is threetimes more common in females. Additionally, to gallbladder pathologies,infections with Salmonella or Helicobacter are common risk factors. GBCis common in South Americans, Indians, Pakistani, Japanese and Koreans,while it is rare in the western world. Genetic changes in GBC are poorlyunderstood. Molecular changes such as p53 mutation, COX2 overexpression,CDKN inactivation, KRAS mutations but also microsatellite instabilityare thought to be involved in GBC carcinogenesis (Kanthan et al., 2015).

As for GBC only 10% of tumors are resectable and even with surgery mostprogress to metastatic disease, prognosis is even worse than for CCCwith a 5-year survival of less than 5%. Although most tumors areunresectable there is still no effective adjuvant therapy (Rakic et al.,2014). Some studies showed that combination of chemotherapeutic drugs orcombination of targeted therapy (anti-VEGFR/EGFR) with chemotherapy ledto an increased overall survival and might be promising treatmentoptions for the future (Kanthan et al., 2015).

Due to the rarity of carcinomas of the biliary tract in general thereare only a few GBC or CCC specific studies, while most of them includeall biliary tract cancers. This is the reason why treatment did notimprove during the last decades and R0 resection still is the onlycurative treatment option.

These unsatisfactory treatment options and low survival rates displaythe need for innovative treatment. There are some clinical studies usingimmunotherapy for the treatment of CCC and GBC. Success was reportedwhen CCC with lymph node metastasis was treated by surgery andpost-operative immunotherapy consisting of CD3-activated T cells andtumor lysate (Higuchi et al., 2006).

For CCC and GBC peptide-based vaccines targeting WT1, NUF2, CDH3,KIF20A, LY6K, TTK, IGF2BP3, or DEPDC1, either as triple/quadrupletherapy or monotherapy combined with gemcitabine, increased overallsurvival about 9-12 months in phase I clinical trials.

Peptide-based vaccines seem to be well tolerated, but only show a modestanti-tumor effect when administered as monotherapy. Dendritic cell-basedvaccines targeting MUC1 or WT1 showed even more promising results.Therapies using cytokine induced killer cell monotherapy or tumorinfiltrating lymphocytes are in phase I/II clinical trials (Marks andYee, 2015).

Considering the severe side-effects and expense associated with treatingcancer, there is a need to identify factors that can be used in thetreatment of cancer in general and gallbladder cancer andcholangiocarcinoma in particular. There is also a need to identifyfactors representing biomarkers for cancer in general and gallbladdercancer and cholangiocarcinoma in particular, leading to better diagnosisof cancer, assessment of prognosis, and prediction of treatment success.

Immunotherapy of cancer represents an option of specific targeting ofcancer cells while minimizing side effects. Cancer immunotherapy makesuse of the existence of tumor associated antigens.

The current classification of tumor associated antigens (TAAs) comprisesthe following major groups:

a) Cancer-testis antigens: The first TAAs ever identified that can berecognized by T cells belong to this class, which was originally calledcancer-testis (CT) antigens because of the expression of its members inhistologically different human tumors and, among normal tissues, only inspermatocytes/spermatogonia of testis and, occasionally, in placenta.Since the cells of testis do not express class I and II HLA molecules,these antigens cannot be recognized by T cells in normal tissues and cantherefore be considered as immunologically tumor-specific. Well-knownexamples for CT antigens are the MAGE family members and NY-ESO-1.b) Differentiation antigens: These TAAs are shared between tumors andthe normal tissue from which the tumor arose. Most of the knowndifferentiation antigens are found in melanomas and normal melanocytes.Many of these melanocyte lineage-related proteins are involved inbiosynthesis of melanin and are therefore not tumor specific butnevertheless are widely used for cancer immunotherapy. Examples include,but are not limited to, tyrosinase and Melan-A/MART-1 for melanoma orPSA for prostate cancer.c) Over-expressed TAAs: Genes encoding widely expressed TAAs have beendetected in histologically different types of tumors as well as in manynormal tissues, generally with lower expression levels. It is possiblethat many of the epitopes processed and potentially presented by normaltissues are below the threshold level for T-cell recognition, whiletheir over-expression in tumor cells can trigger an anticancer responseby breaking previously established tolerance. Prominent examples forthis class of TAAs are Her-2/neu, survivin, telomerase, or WT1.d) Tumor-specific antigens: These unique TAAs arise from mutations ofnormal genes (such as β-catenin, CDK4, etc.). Some of these molecularchanges are associated with neoplastic transformation and/orprogression. Tumor-specific antigens are generally able to induce strongimmune responses without bearing the risk for autoimmune reactionsagainst normal tissues. On the other hand, these TAAs are in most casesonly relevant to the exact tumor on which they were identified and areusually not shared between many individual tumors. Tumor-specificity (or-association) of a peptide may also arise if the peptide originates froma tumor- (-associated) exon in case of proteins with tumor-specific(-associated) isoforms.e) TAAs arising from abnormal post-translational modifications: SuchTAAs may arise from proteins which are neither specific noroverexpressed in tumors but nevertheless become tumor associated byposttranslational processes primarily active in tumors. Examples forthis class arise from altered glycosylation patterns leading to novelepitopes in tumors as for MUC1 or events like protein splicing duringdegradation which may or may not be tumor specific.f) Oncoviral proteins: These TAAs are viral proteins that may play acritical role in the oncogenic process and, because they are foreign(not of human origin), they can evoke a T-cell response. Examples ofsuch proteins are the human papilloma type 16 virus proteins, E6 and E7,which are expressed in cervical carcinoma.

T-cell based immunotherapy targets peptide epitopes derived fromtumor-associated or tumor-specific proteins, which are presented bymolecules of the major histocompatibility complex (MHC). The antigensthat are recognized by the tumor specific T lymphocytes, that is, theepitopes thereof, can be molecules derived from all protein classes,such as enzymes, receptors, transcription factors, etc. which areexpressed and, as compared to unaltered cells of the same origin,usually up-regulated in cells of the respective tumor.

There are two classes of MHC-molecules, MHC class I and MHC class II.MHC class I molecules are composed of an alpha heavy chain andbeta-2-microglobulin, MHC class II molecules of an alpha and a betachain. Their three-dimensional conformation results in a binding groove,which is used for non-covalent interaction with peptides.

MHC class I molecules can be found on most nucleated cells. They presentpeptides that result from proteolytic cleavage of predominantlyendogenous proteins, defective ribosomal products (DRIPs) and largerpeptides. However, peptides derived from endosomal compartments orexogenous sources are also frequently found on MHC class I molecules.This non-classical way of class I presentation is referred to ascross-presentation in the literature (Brossart and Bevan, 1997; Rock etal., 1990). MHC class II molecules can be found predominantly onprofessional antigen presenting cells (APCs), and primarily presentpeptides of exogenous or transmembrane proteins that are taken up byAPCs e.g. during endocytosis, and are subsequently processed.

Complexes of peptide and MHC class I are recognized by CD8-positive Tcells bearing the appropriate T-cell receptor (TCR), whereas complexesof peptide and MHC class II molecules are recognized byCD4-positive-helper-T cells bearing the appropriate TCR. It is wellknown that the TCR, the peptide and the MHC are thereby present in astoichiometric amount of 1:1:1.

CD4-positive helper T cells play an important role in inducing andsustaining effective responses by CD8-positive cytotoxic T cells. Theidentification of CD4-positive T-cell epitopes derived from tumorassociated antigens (TAA) is of immense importance for the developmentof pharmaceutical products for triggering anti-tumor immune responses(Gnjatic et al., 2003). At the tumor site, T helper cells, support acytotoxic T cell− (CTL−) friendly cytokine milieu (Mortara et al., 2006)and attract effector cells, e.g. CTLs, natural killer (NK) cells,macrophages, and granulocytes (Hwang et al., 2007).

In the absence of inflammation, expression of MHC class II molecules ismainly restricted to cells of the immune system, especially professionalantigen-presenting cells (APC), e.g., monocytes, monocyte-derived cells,macrophages, dendritic cells. In cancer patients, cells of the tumorhave been found to express MHC class II molecules (Dengjel et al.,2006).

Elongated (longer) peptides of the invention can act as MHC class IIactive epitopes.

T-helper cells, activated by MHC class II epitopes, play an importantrole in orchestrating the effector function of CTLs in anti-tumorimmunity. T-helper cell epitopes that trigger a T-helper cell responseof the TH1 type support effector functions of CD8-positive killer Tcells, which include cytotoxic functions directed against tumor cellsdisplaying tumor-associated peptide/MHC complexes on their cellsurfaces. In this way tumor-associated T-helper cell peptide epitopes,alone or in combination with other tumor-associated peptides, can serveas active pharmaceutical ingredients of vaccine compositions thatstimulate anti-tumor immune responses.

It was shown in mammalian animal models, e.g., mice, that even in theabsence of CD8-positive T lymphocytes, CD4-positive T cells aresufficient for inhibiting manifestation of tumors via inhibition ofangiogenesis by secretion of interferon-gamma (IFNγ) (Beatty andPaterson, 2001; Mumberg et al., 1999). There is evidence for CD4 T cellsas direct anti-tumor effectors (Braumuller et al., 2013; Tran et al.,2014).

Since the constitutive expression of HLA class II molecules is usuallylimited to immune cells, the possibility of isolating class II peptidesdirectly from primary tumors was previously not considered possible.However, Dengjel et al. were successful in identifying a number of MHCClass II epitopes directly from tumors (WO 2007/028574, EP 1 760 088B1).

Since both types of response, CD8 and CD4 dependent, contribute jointlyand synergistically to the anti-tumor effect, the identification andcharacterization of tumor-associated antigens recognized by either CD8+T cells (ligand: MHC class I molecule+peptide epitope) or byCD4-positive T-helper cells (ligand: MHC class II molecule+peptideepitope) is important in the development of tumor vaccines.

For an MHC class I peptide to trigger (elicit) a cellular immuneresponse, it also must bind to an MHC-molecule. This process isdependent on the allele of the MHC-molecule and specific polymorphismsof the amino acid sequence of the peptide. MHC-class-I-binding peptidesare usually 8-14, or 8-12, or 8-11 or 8-10 amino acid residues in lengthand usually contain two conserved residues (“anchors”) in their sequencethat interact with the corresponding binding groove of the MHC-molecule.In this way, each MHC allele has a “binding motif” determining whichpeptides can bind specifically to the binding groove.

In the MHC class I dependent immune reaction, peptides not only have tobe able to bind to certain MHC class I molecules expressed by tumorcells, they subsequently also have to be recognized by T cells bearingspecific T cell receptors (TCR).

For proteins to be recognized by T-lymphocytes as tumor-specific or-associated antigens, and to be used in a therapy, particularprerequisites must be fulfilled. The antigen should be expressed mainlyby tumor cells and not, or in comparably small amounts, by normalhealthy tissues. In a preferred embodiment, the peptide should beover-presented by tumor cells as compared to normal healthy tissues. Itis furthermore desirable that the respective antigen is not only presentin a type of tumor, but also in high concentrations (i.e. copy numbersof the respective peptide per cell). Tumor-specific and tumor-associatedantigens are often derived from proteins directly involved intransformation of a normal cell to a tumor cell due to their function,e.g. in cell cycle control or suppression of apoptosis. Additionally,downstream targets of the proteins directly causative for atransformation may be up-regulated und thus may be indirectlytumor-associated. Such indirect tumor-associated antigens may also betargets of a vaccination approach (Singh-Jasuja et al., 2004). It isessential that epitopes are present in the amino acid sequence of theantigen, in order to ensure that such a peptide (“immunogenic peptide”),being derived from a tumor associated antigen, leads to an in vitro orin vivo T-cell-response.

Basically, any peptide able to bind an MHC molecule may function as aT-cell epitope. A prerequisite for the induction of an in vitro or invivo T-cell-response is the presence of a T cell having a correspondingTCR and the absence of immunological tolerance for this particularepitope.

Therefore, TAAs are a starting point for the development of a T cellbased therapy including but not limited to tumor vaccines. The methodsfor identifying and characterizing the TAAs are usually based on the useof T-cells that can be isolated from patients or healthy subjects, orthey are based on the generation of differential transcription profilesor differential peptide expression patterns between tumors and normaltissues. However, the identification of genes over-expressed in tumortissues or human tumor cell lines, or selectively expressed in suchtissues or cell lines, does not provide precise information as to theuse of the antigens being transcribed from these genes in an immunetherapy.

This is because only an individual subpopulation of epitopes of theseantigens are suitable for such an application since a T cell with acorresponding TCR has to be present and the immunological tolerance forthis particular epitope needs to be absent or minimal.

In a very preferred embodiment of the invention it is thereforeimportant to select only those over- or selectively presented peptidesagainst which a functional and/or a proliferating T cell can be found.Such a functional T cell is defined as a T cell, which upon stimulationwith a specific antigen can be clonally expanded and is able to executeeffector functions (“effector T cell”).

In case of targeting peptide-MHC by specific TCRs (e.g. soluble TCRs)and antibodies or other binding molecules (scaffolds) according to theinvention, the immunogenicity of the underlying peptides is secondary.In these cases, the presentation is the determining factor.

SUMMARY OF THE INVENTION

In a first aspect of the present invention, the present inventionrelates to a peptide comprising an amino acid sequence selected from thegroup consisting of SEQ ID NO: 1 to SEQ ID NO: 32 or a variant sequencethereof which is at least 77%, preferably at least 88%, homologous(preferably at least 77% or at least 88% identical) to SEQ ID NO: 1 toSEQ ID NO: 32, wherein said variant binds to MHC and/or induces T cellscross-reacting with said peptide, or a pharmaceutical acceptable saltthereof, wherein said peptide is not the underlying full-lengthpolypeptide.

The present invention further relates to a peptide of the presentinvention comprising a sequence that is selected from the groupconsisting of SEQ ID NO: 1 to SEQ ID NO: 32 or a variant thereof, whichis at least 77%, preferably at least 88%, homologous (preferably atleast 77% or at least 88% identical) to SEQ ID NO: 1 to SEQ ID NO: 32,wherein said peptide or variant thereof has an overall length of between8 and 100, preferably between 8 and 30, and most preferred of between 8and 14 amino acids.

The following tables show the peptides according to the presentinvention, their respective SEQ ID NOs, and the prospective source(underlying) genes for these peptides. All peptides in Table 1 and Table2 bind to HLA-A*02. The peptides in Table 2 have been disclosed beforein large listings as results of high-throughput screenings with higherror rates or calculated using algorithms, but have not been associatedwith cancer at all before. The peptides in Table 3 are additionalpeptides that may be useful in combination with the other peptides ofthe invention. The peptides in Tables 4A and B are furthermore useful inthe diagnosis and/or treatment of various other malignancies thatinvolve an over-expression or over-presentation of the respectiveunderlying polypeptide.

TABLE 1 Peptides according to the present invention. SEQ IDOfficial Gene No. Sequence GeneID(s) Symbol(s) 1 YAAEIASAL 6446, 23678,SGK1, SGK3, 100533105 C8orf44-SGK3 2 AAYPEIVAV 348654 GEN1 3 EMDSTVITV 26137 ZBTB20 4 FLLEAQNYL 149281 METTL11B 5 GLIDEVMVLL  54905 CYP2W1 6LLLPLLPPLSPS 347252 IGFBPL1 7 LLLSDPDKVTI 3700, 375346 ITIH4, TMEM110 8LSASLVRIL  55655 NLRP2 9 RLAKLTAAV 283209 PGM2L1 10 SAFPFPVTVSL  79939SLC35E1 11 SIIDFTVTM   1767 DNAH5 12 TILPGNLQSW 80317, 387032ZKSCAN3, ZKSCAN4 13 VLPRAFTYV   5314 PKHD1 14 YGIEFVVGV  56670 SUCNR1 15SVIDSLPEI  79830 ZMYM1 16 AVMTDLPVI  23041 MON2 17 VLYDNTQLQL 389072PLEKHM3 18 SLSPDLSQV   2153 F5 19 TAYPQVVVV  57494 RIMKLB 20 VLQDELPQL  1953 MEGF6 21 IAFPTSISV   5036 PA2G4 22 SAFGFPVIL  54741 LEPROT 23SLLSELLGV  11135 CDC42EP1

TABLE 2 Additional peptides according to the presentinvention with no prior known cancer association. SEQ ID Official GeneNo. Sequence GeneID(s) Symbol(s) 24 ISAPLVKTL    994 CDC25B 25NLSETASTMAL  25897 RNF19A 26 TAQTLVRIL   3608 ILF2 27 ALAEQVQKA  79078C1orf50 28 YASGSSASL   5339 PLEC 29 FASEVSNVL   8027 STAM 30 FASGLIHRV200185 KRTCAP2 31 IAIPFLIKL  26090 ABHD12 32 YVISQVFEI 10447, 51384FAM3C, WNT16

TABLE 3 Peptides of the invention useful for e.g.personalized cancer therapies. SEQ ID Official Gene No. SequenceGeneID(s) Symbol(s) 33 ILGTEDLIVEV 79719 AAGAB 34 LLWGNLPEI729533, 653820 FAM72A, FAM72B 35 GLIDEVMVL 54905 CYP2W1 36 ILVDWLVQV 9133 CCNB2 37 KIQEMQHFL  4321 MMP12 38 KIQEILTQV 10643 IGF2BP3

The present invention furthermore generally relates to the peptidesaccording to the present invention for use in the treatment ofproliferative diseases, such as, for example, acute myeloid leukemia,melanoma, small cell lung cancer, non-small cell lung cancer,non-Hodgkin lymphoma, chronic lymphocytic leukemia, pancreatic cancer,liver cancer, ovarian cancer, head and neck cancer, urinary bladdercancer, breast cancer, and kidney cancer.

Particularly preferred are the peptides—alone or incombination—according to the present invention selected from the groupconsisting of SEQ ID NO: 1 to SEQ ID NO: 32. More preferred are thepeptides—alone or in combination—selected from the group consisting ofSEQ ID NO: 1 to SEQ ID NO: 16 (see Table 1), and their uses in theimmunotherapy of gallbladder cancer and cholangiocarcinoma, acutemyeloid leukemia, melanoma, small cell lung cancer, non-small cell lungcancer, non-Hodgkin lymphoma, chronic lymphocytic leukemia, pancreaticcancer, liver cancer, ovarian cancer, head and neck cancer, urinarybladder cancer, breast cancer, and kidney cancer, and preferablygallbladder cancer and cholangiocarcinoma. As shown in the followingTables 4A and B, many of the peptides according to the present inventionare also found on other tumor types and can, thus, also be used in theimmunotherapy of other indications. Also refer to FIGS. 1A to A-1G andExample 1.

TABLE 4A Peptides according to the present invention andtheir specific uses in other proliferativediseases, especially in other cancerous diseases.The table shows for selected peptides on whichadditional tumor types they were found and eitherover-presented on more than 5% of the measuredtumor samples, or presented on more than 5% ofthe measured tumor samples with a ratio ofgeometric means tumor vs normal tissues beinglarger than 3. Over-presentation is defined ashigher presentation on the tumor sample ascompared to the normal sample with highestpresentation. Normal tissues against which over-presentation was tested were: adipose tissue,adrenal gland, artery, bone marrow, brain, centralnerve, colon, duodenum, esophagus, eye,gallbladder, heart, kidney, liver, lung, lymphnode, mononuclear white blood cells, pancreas,parathyroid gland, peripheral nerve, peritoneum,pituitary, pleura, rectum, salivary gland,skeletal muscle, skin, small intestine, spleen,stomach, thyroid gland, trachea, ureter, urinary bladder, vein. SEQ IDOther relevant organs/ No. Sequence diseases 1 YAAEIASAL AML, Melanoma 6LLLPLLPPLSPS SCLC,PC 7 LLLSDPDKVTI HCC 16 AVMTDLPVI CLL, NHL, AML 17VLYDNTQLQL NHL, AML 18 SLSPDLSQV HCC, NHL, AML 20 VLQDELPQLNSCLC, NHL, AML, HNSCC, OC 21 IAFPTSISV BRCA 25 NLSETASTMALSCLC, NHL, Urinary bladder cancer 26 TAQTLVRIL CLL, BRCA 28 YASGSSASLRCC, AML, Melanoma 29 FASEVSNVL SCLC, RCC, CLL, AML, Melanoma 30FASGLIHRV CLL, AML 31 IAIPFLIKL SCLC, NHL 32 YVISQVFEI CLL, MelanomaNSCLC = non-small cell lung cancer, SCLC = small cell lung cancer, RCC= kidney cancer, HCC = liver cancer, PC = pancreatic cancer, BRCA= breast cancer, CLL = chronic lymphocytic leukemia, AML = acute myeloidleukemia, NHL = non-Hodgkin lymphoma, OC = ovarian cancer, HNSCC = headand neck cancer.

TABLE 4B Peptides according to the present inventionand their specific uses in other proliferativediseases, especially in other cancerousdiseases. The table shows for selected peptideson which additional tumor types they were foundand either over-presented on more than 5% ofthe measured tumor samples, or presented on morethan 5% of the measured tumor samples with aratio of geometric means tumor vs normal tissuesbeing larger than three. Over-presentation isdefined as higher presentation on the tumorsample as compared to the normal sample withhighest presentation. Normal tissues againstwhich over-presentation was tested were:adipose tissue, adrenal gland, artery, bonemarrow, brain, central nerve, colon, esophagus,eye, gallbladder, heart, kidney, liver, lung,lymph node, white blood cells, pancreas,parathyroid gland, peripheral nerve, peritoneum,pituitary, pleura, rectum, salivary gland,skeletal muscle, skin, small intestine, spleen,stomach, thymus, thyroid gland, trachea, ureter, urinary bladder, vein.SEQ ID No Sequence Additional Entities 6 LLLPLLPPLSPS Brain Cancer 17VLYDNTQLQL CLL, OC, Urinary bladder cancer 24 ISAPLVKTL CLL 31 IAIPFLIKLHCC, BRCA 32 YVISQVFEI NSCLC NSCLC = non-small cell lung cancer, HCC= liver cancer, BRCA = breast cancer, CLL = chronic lymphocyticleukemia, OC = ovarian cancer.

Thus, another aspect of the present invention relates to the use of atleast one peptide according to the present invention according to anyone of SEQ ID No. 1, 16, 17, 18, 20, 28, 29, and 30 for the—in onepreferred embodiment combined—treatment of acute myeloid leukemia.

Thus, another aspect of the present invention relates to the use of atleast one peptide according to the present invention according to anyone of SEQ ID No. 1, 28, 29, and 32 for the—in one preferred embodimentcombined—treatment of melanoma.

Thus, another aspect of the present invention relates to the use of atleast one peptide according to the present invention according to anyone of SEQ ID No. 6, 25, 29, and 31 for the—in one preferred embodimentcombined—treatment of small cell lung cancer.

Thus, another aspect of the present invention relates to the use of atleast one peptide according to the present invention according to anyone of SEQ ID No. 32, and 20 for the—in one preferred embodimentcombined—treatment of non-small cell lung cancer.

Thus, another aspect of the present invention relates to the use of atleast one peptide according to the present invention according to anyone of SEQ ID No. 16, 17, 18, 20, 25, and 31 for the—in one preferredembodiment combined—treatment of non-Hodgkin lymphoma.

Thus, another aspect of the present invention relates to the use of atleast one peptide according to the present invention according to anyone of SEQ ID No. 16, 26, 29, 30, and 32 for the—in one preferredembodiment combined—treatment of chronic lymphocytic leukemia.

Thus, another aspect of the present invention relates to the use of atleast one peptide according to the present invention according to anyone of SEQ ID No. 6 for the—in one preferred embodimentcombined—treatment of pancreatic cancer.

Thus, another aspect of the present invention relates to the use of atleast one peptide according to the present invention according to anyone of SEQ ID No. 7, 31, and 18 for the—in one preferred embodimentcombined—treatment of liver cancer.

Thus, another aspect of the present invention relates to the use of atleast one peptide according to the present invention according to anyone of SEQ ID No. 17, and 20 for the—in one preferred embodimentcombined—treatment of ovarian cancer.

Thus, another aspect of the present invention relates to the use of atleast one peptide according to the present invention according to anyone of SEQ ID No. 20 for the—in one preferred embodimentcombined—treatment of head and neck cancer.

Thus, another aspect of the present invention relates to the use of atleast one peptide according to the present invention according to anyone of SEQ ID No. 17, and 25 for the—in one preferred embodimentcombined—treatment of urinary bladder cancer.

Thus, another aspect of the present invention relates to the use of atleast one peptide according to the present invention according to anyone of SEQ ID No. 21, 31, and 26 for the—in one preferred embodimentcombined—treatment of breast cancer.

Thus, another aspect of the present invention relates to the use of atleast one peptide according to the present invention according to anyone of SEQ ID No. 28, and 29 for the—in one preferred embodimentcombined—treatment of kidney cancer.

Thus, another aspect of the present invention relates to the use of atleast one peptide according to the present invention according to anyone of SEQ ID No. 17, and 24 for the—in one preferred embodimentcombined—treatment of CLL.

Thus, another aspect of the present invention relates to the use of thepeptides according to the present invention for the—preferablycombined—treatment of a proliferative disease selected from the group ofgallbladder cancer and cholangiocarcinoma, acute myeloid leukemia,melanoma, small cell lung cancer, non-small cell lung cancer,non-Hodgkin lymphoma, chronic lymphocytic leukemia, pancreatic cancer,liver cancer, ovarian cancer, head and neck cancer, urinary bladdercancer, breast cancer, and kidney cancer.

The present invention furthermore relates to peptides according to thepresent invention that have the ability to bind to a molecule of thehuman major histocompatibility complex (MHC) class-I or—in an elongatedform, such as a length-variant—MHC class-II.

The present invention further relates to the peptides according to thepresent invention wherein said peptides (each) consist of or consistessentially of an amino acid sequence according to SEQ ID NO: 1 to SEQID NO: 32.

The present invention further relates to the peptides according to thepresent invention, wherein said peptide is modified and/or includesnon-peptide bonds.

The present invention further relates to the peptides according to thepresent invention, wherein said peptide is part of a fusion protein, inparticular fused to the N-terminal amino acids of the HLA-DRantigen-associated invariant chain (li), or fused to (or into thesequence of) an antibody, such as, for example, an antibody that isspecific for proteins presented by dendritic cells.

The present invention further relates to a nucleic acid, encoding thepeptides according to the present invention. The present inventionfurther relates to the nucleic acid according to the present inventionthat is DNA, cDNA, PNA, RNA or combinations thereof.

The present invention further relates to an expression vector capable ofexpressing and/or expressing a nucleic acid according to the presentinvention.

The present invention further relates to a peptide according to thepresent invention, a nucleic acid according to the present invention oran expression vector according to the present invention for use in thetreatment of diseases and in medicine, in particular in the treatment ofcancer.

The present invention further relates to antibodies that are specificagainst the peptides according to the present invention or complexes ofsaid peptides according to the present invention with MHC, and methodsof making these.

The present invention further relates to T-cell receptors (TCRs), inparticular soluble TCR (sTCRs) and cloned TCRs engineered intoautologous or allogeneic T cells, and methods of making these, as wellas NK cells or other cells bearing said TCR or cross-reacting with saidTCRs.

The antibodies and TCRs are additional embodiments of theimmunotherapeutic use of the peptides according to the invention athand.

The present invention further relates to a host cell comprising anucleic acid according to the present invention or an expression vectoras described before. The present invention further relates to the hostcell according to the present invention that is an antigen presentingcell, and preferably is a dendritic cell.

The present invention further relates to a method for producing apeptide according to the present invention, said method comprisingculturing the host cell according to the present invention, andisolating the peptide from said host cell or its culture medium.

The present invention further relates to said method according to thepresent invention, wherein the antigen is loaded onto class I or II MHCmolecules expressed on the surface of a suitable antigen-presenting cellor artificial antigen-presenting cell by contacting a sufficient amountof the antigen with an antigen-presenting cell.

The present invention further relates to the method according to thepresent invention, wherein the antigen-presenting cell comprises anexpression vector capable of expressing or expressing said peptidecontaining SEQ ID No. 1 to SEQ ID No.: 32, preferably containing SEQ IDNo. 1 to SEQ ID No. 16, or a variant amino acid sequence.

The present invention further relates to activated T cells, produced bythe method according to the present invention, wherein said T cellselectively recognizes a cell which expresses a polypeptide comprisingan amino acid sequence according to the present invention.

The present invention further relates to a method of killing targetcells in a patient which target cells aberrantly express a polypeptidecomprising any amino acid sequence according to the present invention,the method comprising administering to the patient an effective numberof T cells as produced according to the present invention.

The present invention further relates to the use of any peptide asdescribed herein, the nucleic acid according to the present invention,the expression vector according to the present invention, the cellaccording to the present invention, the activated T lymphocyte, the Tcell receptor or the antibody or other peptide- and/orpeptide-MHC-binding molecules according to the present invention as amedicament or in the manufacture of a medicament. Preferably, saidmedicament is active against cancer.

Preferably, said medicament is a cellular therapy, a vaccine or aprotein based on a soluble TCR or antibody.

The present invention further relates to a use according to the presentinvention, wherein said cancer cells are gallbladder cancer andcholangiocarcinoma, acute myeloid leukemia, melanoma, small cell lungcancer, non-small cell lung cancer, non-Hodgkin lymphoma, chroniclymphocytic leukemia, pancreatic cancer, liver cancer, ovarian cancer,head and neck cancer, urinary bladder cancer, breast cancer, and kidneycancer, and preferably gallbladder cancer and cholangiocarcinoma cells.

The present invention further relates to biomarkers based on thepeptides according to the present invention, herein called “targets”that can be used in the diagnosis of cancer, preferably gallbladdercancer and cholangiocarcinoma. The marker can be over-presentation ofthe peptide(s) themselves, or over-expression of the correspondinggene(s). The markers may also be used to predict the probability ofsuccess of a treatment, preferably an immunotherapy, and most preferredan immunotherapy targeting the same target that is identified by thebiomarker. For example, an antibody or soluble TCR can be used to stainsections of the tumor to detect the presence of a peptide of interest incomplex with MHC.

Optionally the antibody carries a further effector function such as animmune stimulating domain or toxin.

The present invention also relates to the use of these novel targets inthe context of cancer treatment.

Both therapeutic and diagnostic uses against additional cancerousdiseases are disclosed in the following more detailed description of theunderlying expression products (polypeptides) of the peptides accordingto the invention.

CDC25B is known to be a downstream target of the oncogenic transcriptionfactor FoxM1. FoxM1 and its downstream target effectors aredown-regulated in gastric cancer, gliomas, cholangiocarcinoma, and acutemyeloid leukemia (Zhang et al., 2014a; Chan-On et al., 2015; Niu et al.,2015; Li et al., 2016). MicroRNA-211 was shown to be a direct negativeregulator of CDC25B in triple-negative breast cancer cells. The loss ofmiRNA-211 and the resulting increase of CDC25B expression lead toincreased genomic instability (Song and Zhao, 2015). CDC25B was shown tobe up-regulated in gastric cancer cells by YWHAE silencing inducing cellproliferation, invasion and migration (Leal et al., 2016). CDC25B wasshown to be down-regulated by the bromodomain inhibitor JQ1 whichsuppresses growth of pancreatic ductal adenocarcinoma in patient-derivedxenograft models (Garcia et al., 2016). CDC25B was shown to bedown-regulated in small intestinal neuroendocrine tumors (Kim et al.,2016b).

CYP2W1 is over-expressed in a variety of human cancers includinghepatocellular, colorectal and gastric cancer. CYP2W1 over-expression isassociated with tumor progression and poor survival (Aung et al., 2006;Gomez et al., 2010; Zhang et al., 2014b). Due to tumor-specificexpression, CYP2W1 is an interesting drug target or enzymatic activatorof pro-drugs during cancer therapy (Karigren and Ingelman-Sundberg,2007; Nishida et al., 2010).

The DNAH5 gene was reported to be recurrently mutated in myeloma and itsexpression was shown to be commonly dysregulated in colorectal cancer(Walker et al., 2012; Xiao et al., 2015).

It was shown that venous thromboembolism (VTE) occurs frequently incancer patients.

A combination of F5 variants that are associated with VTE and cancersynergistically increases the risk of VTE (Gran et al., 2016). Theprocoagulant state in cancer increases the thrombotic risk, but alsosupports tumor progression. Four SNPs in F5 were shown to be associatedwith breast cancer. Therefore, targeting the coagulation processes incancer is of high importance (Tinholt et al., 2014). It was shown thatF5 mutation is a risk factor for thromboemboli occurrence in childrenwith acute lymphoblastic leukemia (Sivaslioglu et al., 2014). F5 wasshown to be a candidate serum biomarker for prostate adenocarcinoma(Klee et al., 2012).

A change in expression of FAMC3 has been noted in pancreaticcancer-derived cells (RefSeq, 2002). In melanoma, FAMC3 has beenidentified as a candidate biomarker for autophagy, an important tumorcell survival mechanism (Kraya et al., 2015). FAMC3 plays an essentialrole in the epithelial-mesenchymal transition which correlates withaggressiveness, metastatic progression of tumors and poor survivalespecially in hepatocellular cancer, colorectal cancer, lung and breastcancers (Csiszar et al., 2014; Gao et al., 2014; Song et al., 2014;Chaudhury et al., 2010; Lahsnig et al., 2009).

Mutations in GEN1 have been reported to be associated with breast cancerrisk, but other studies could not confirm the role of GEN1 as a breastcancer predisposition gene (Kuligina et al., 2013; Sun et al., 2014;Turnbull et al., 2010).

IGFBPL1 is a regulator of insulin-growth factors and is down-regulatedin breast cancer cell lines by aberrant hypermethylation. Methylation inIGFBPL1 was clearly associated with worse overall survival anddisease-free survival (Smith et al., 2007).

ILF2, also known as NF45, encodes a transcription factor required forT-cell expression of the interleukin 2 gene (RefSeq, 2002). ILF2 wasshown to be up-regulated in hepatocellular carcinoma, pancreatic ductaladenocarcinoma and non-small cell lung cancer (Ni et al., 2015; Wan etal., 2015; Cheng et al., 2016). Expression of ILF2 in liver cancer cellswas described as being associated with the regulation of cell growth andapoptosis via regulation of Bcl-2, Bok, BAX, and cIAP1 (Cheng et al.,2016). Expression of ILF2 was shown to correlate with tumor size,histological differentiation and TNM stage, while over-expression ofILF2 was shown to be associated with poor prognosis of pancreatic ductaladenocarcinoma. The differentiated expression of ILF2 in pancreaticductal adenocarcinoma cell cultures showed effects on cell cycleprogression (Wan et al., 2015). Up-regulated expression of ILF2 innon-small cell lung cancer was shown to be associated with tumor cellproliferation and poor prognosis (Ni et al., 2015).

ITIH4 is a member of the ITI family of plasma protease inhibitors thatcontribute to extracellular matrix stability by covalent linkage tohyaluronan. ITIH4 was down-regulated in several tumor tissues includingcolon, stomach, ovary, lung, kidney, rectum and prostate (Hamm et al.,2008). Serum ITIH4 levels are reduced in HCC patients compared to thatin chronic hepatitis B and cirrhosis patients, and low serum ITIH4levels are associated with shorter survival in HBV-associated HCCpatients (Noh et al., 2014).

KRTCAP2 encodes keratinocyte associated protein 2 and is localized onchromosome 1q22 (RefSeq, 2002). Studies uncovered a cancer-enrichedchimeric RNA as the result of splicing between MUC1, TRIM46, and KRTCAP2in high-grade serous ovarian cancer (HGSC) cells, which might be used asa clinical biomarker and therapeutic target (Kannan et al., 2015).

MEGF6, also known as EGFL3, encodes the multiple EGF like domains 6protein and is located on chromosome 1p36.3 (RefSeq, 2002). MEGF6 wasdescribed as a hepatocellular carcinoma-related gene which shows severalpolymorphisms in the tissues of hepatocellular carcinomas (Wang et al.,2005).

NLRP2 (also known as NALP2) encodes the NLR family, pyrin domaincontaining 2 protein and is involved in the activation of caspase-1 andmay also form protein complexes activating proinflammatory caspases.NLRP7 is a paralog of NLRP2 (RefSeq, 2002; Wu et al., 2010; Slim et al.,2012). The PYRIN domain of NLRP2 inhibits cell proliferation and tumorgrowth of glioblastoma (Wu et al., 2010). An ATM/NLRP2/MDC1-dependentpathway may shut down ribosomal gene transcription in response tochromosome breaks (Kruhlak et al., 2007). Mutations in NLRP2 can causerare human imprinting disorders such as familial hydatidiform mole,Beckwith-Wiedemann syndrome and familial transient neonatal diabetesmellitus (Aghajanova et al., 2015; Dias and Maher, 2013; Ulker et al.,2013). NLRP2 inhibits NF-kappaB activation (Kinoshita et al., 2005;Kinoshita et al., 2006; Fontalba et al., 2007; Bruey et al., 2004).

PA2G4 encodes proliferation-associated 2G, an RNA-binding protein thatis involved in growth regulation and might be involved in ribosomeassembly. It has been implicated in induction of differentiation ofhuman cancer cells (RefSeq, 2002). PA2G4 was identified to bedown-regulated in esophageal squamous cell carcinoma. Over-expression ofPA2G4 inhibited the tumorigenesis and growth of the cells and inducedapoptosis. These results indicate that PA2G4 may suppress the growth ofesophageal carcinoma cells (Jiang et al., 2016). PA2G4 was shown to beup-regulated in cervical cancer tissues and might be combined with p53expression levels an effective predictor of metastatic potential andpatient prognosis (Liu et al., 2015c; Liu et al., 2015b). PA2G4 wasshown to be up-regulated in pancreatic ductal adenocarcinoma and couldserve as a prognostic indicator and potential target (Gong et al.,2015). Forced PA2G4 expression was shown to suppress growth, migrationand invasion in thyroid cancer cells by up-regulating a majortumor-suppressor RASAL (Liu et al., 2015a). PA2G4 has been reported tobe down-regulated in hepatocellular carcinoma (HCC) and might serve as aprognostic marker and promising therapeutic target of HCC (Hu et al.,2014).

PKHD1 encodes polycystic kidney and hepatic disease 1. Mutations in thisgene cause autosomal recessive polycystic kidney disease (RefSeq, 2002).PKHD1 was seen to have loss of function mutations in anaplastic thyroidcarcinoma (Jeon et al., 2016).

PLEC encodes the plakin family member plectin, a protein involved in thecross-linking and organization of the cytoskeleton and adhesioncomplexes (Bouameur et al., 2014). PLEC is over-expressed in colorectaladenocarcinoma, head and neck squamous cell carcinoma and pancreaticcancer (Lee et al., 2004; Katada et al., 2012; Bausch et al., 2011).

RNF19A encodes ring finger protein 19A, RBR E3 ubiquitin protein ligase.The encoded protein may be involved in amyotrophic lateral sclerosis andParkinson's disease (RefSeq, 2002). RNF19A mRNA levels were shown to be2-fold higher in the blood of patients with prostate cancer than inhealthy controls. Therefore, RNF19A might be a relevant biomarker forprostate cancer detection (Bai et al., 2012). RNF19A was identifiedbeing differentially expressed in cancer-associated fibroblasts that areimportant for cancer development and progression (Bozoky et al., 2013).

SGK1 encodes a serine/threonine protein kinase that plays an importantrole in cellular stress response. It activates certain potassium,sodium, and chloride channels, suggesting an involvement in theregulation of processes such as cell survival, neuronal excitability,and renal sodium excretion. High levels of expression may contribute toconditions such as hypertension and diabetic nephropathy. SGK1 can beactivated by insulin and growth factors via PI3K and PDK1 (RefSeq,2002). SGK1 expression is rapidly up-regulated by glucocorticoidadministration which may decrease chemotherapy effectiveness in ovariancancer. In turn, the isoflavinoid Genistein has been found to have aninhibitory effect on colorectal cancer by reducing SGK1 expression(Melhem et al., 2009; Qin et al., 2015). Increased SGK1 expression hasbeen found in several human tumors, including prostate carcinoma,non-small cell lung cancer and hepatocellular carcinoma. SGK1 hasanti-apoptotic properties and regulates cell survival, proliferation anddifferentiation via phosphorylation of MDM2, which leads to theubiquitination and proteasomal degradation of p53. Direct SGK1inhibition can be effective in hepatic cancer therapy, either alone orin combination with radiotherapy (Lang et al., 2010; Abbruzzese et al.,2012; Isikbay et al., 2014; Talarico et al., 2015).

SGK3 encodes a phosphoprotein of the Ser/Thr protein kinase family whichphosphorylates several target proteins and has a role in neutral aminoacid transport and activation of potassium and chloride channels(RefSeq, 2002). SGK3 function was shown to be associated with theoncogenic driver INPP4B in colon cancer and in breast cancer (Gasser etal., 2014; Guo et al., 2015). SGK3 was described as a down-streammediator of phosphatidylinositol 3-kinase oncogenic signaling whichmediates pivotal roles in oncogenic progress in various cancers,including breast cancer, ovarian cancer and hepatocellular carcinoma(Hou et al., 2015). SGK3 was described to serve as a hallmarkinteracting with numerous molecules in cell proliferation, growth,migration and tumor angiogenesis (Hou et al., 2015). SGK3 was shown topromote hepatocellular carcinoma growth and survival throughinactivating glycogen synthase kinase 3 beta and Bcl-2-associated deathpromoter, respectively (Liu et al., 2012). SGK3 was shown to beassociated with poor outcome in hepatocellular carcinoma patients (Liuet al., 2012). Thus, SGK3 may provide a prognostic biomarker forhepatocellular carcinoma outcome prediction and a novel therapeutictarget (Liu et al., 2012). SGK3 was described as an important mediatorof PDK1 activities in melanoma cells which contributes to the growth ofBRAF-mutant melanomas and may be a potential therapeutic target(Scortegagna et al., 2015). SGK3 was described as an androgen receptortranscriptional target that promotes prostate cell proliferation throughactivation of p70 S6 kinase and up-regulation of cyclin D1 (Wang et al.,2014). Knock-down of SGK3 was shown to decrease LNCaP prostate cancercell proliferation by inhibiting G1 to S phase cell cycle progression(Wang et al., 2014). SGK3 was shown to be associated with estrogenreceptor expression in breast cancer and its expression was shown to bepositively correlated with tumor prognosis (Xu et al., 2012).

SLC35E1 encodes the protein solute carrier family 35, member E1 and islocated on chromosome 19p13.11 (RefSeq, 2002). SLC35E1 was shown to beassociated with rectal carcinoma response to neoadjuvantradiochemotherapy (Rimkus et al., 2008).

STAM encodes a member of the signal-transducing adaptor molecule familythat mediates down-stream signaling of cytokine receptors and also playsa role in ER to Golgi trafficking. STAM associates with hepatocytegrowth factor-regulated substrates to form the endosomal sorting complexrequired for transport-0 (ESCRT-0), which sorts ubiquitinated membraneproteins to the ESCRT-1 complex for lysosomal degradation (RefSeq,2002). STAM has been found to be over-expressed in locally advancedcervical cancer and in tumors in young patients with spinal ependymomas(Korshunov et al., 2003; Campos-Parra et al., 2016). STAM is adownstream target of ZNF331, a gene down-regulated in gastric cancer,which then leads to down-regulation of STAM as well (Yu et al., 2013).STAM has been associated with the unfavorable 11q deletion in chroniclymphocytic leukemia (Aalto et al., 2001).

WNT16, wingless-type MMTV integration site family, member 16 encodes asecreted signaling protein which is implicated in oncogenesis and inseveral developmental processes, including regulation of cell fate andpatterning during embryogenesis (RefSeq, 2002). The expression of WNT16was shown to be up-regulated in t(1;19) chromosomaltranslocation-containing acute lymphoblastoid leukemia (ALL) and play animportant role in leukemogenesis (Casagrande et al., 2006; Mazieres etal., 2005). A study of ALL cell lines and samples from patients with ALLshowed that the up-regulation of WNT16 and few other Wnt target geneswas caused by the methylation of Wnt inhibitors which was furtherassociated with significantly decreased 10-year disease-free survivaland overall survival (Roman-Gomez et al., 2007).

ZBTB20 encodes zinc finger and BTB domain containing 20 and is locatedon chromosome 3q13.2 (RefSeq, 2002). ZBTB20 promotes cell proliferationin non-small cell lung cancer through repression of FoxO1 (Zhao et al.,2014). ZBTB20 expression is increased in hepatocellular carcinoma andassociated with poor prognosis (Wang et al., 2011). Polymorphism inZBTB20 gene is associated with gastric cancer (Song et al., 2013).

ZKSCAN3 encodes zinc finger with KRAB and SCAN domains 3 and is locatedon chromosome 6p22.1 (RefSeq, 2002). ZKSCAN3 is up-regulated in invasivecolonic tumor cells and their liver metastases. ZKSCAN3 is expressed ina majority of prostate cancer samples, but not in normal prostatetissues. ZKSCAN3 gene amplification was observed in metastatic prostatecancers and lymph node metastases but not in primary prostate cancers.ZKSCAN3 plays a critical role in promoting prostate cancer cellmigration (Zhang et al., 2012). ZKSCAN3 is a driver of colon cancerprogression which regulates the expression of several genes favoringtumor progression (Yang et al., 2008). ZKSCAN3 mutation contributes tomyelomagenesis as well as transformation from myeloma to overtextramedullary disease such as secondary plasma cell leukemia (Egan etal., 2012).

ZKSCAN3 suppression reduces cyclin D2 levels and inhibits myeloma cellline proliferation. ZKSCAN3 over-expression induces cyclin D2 in myelomacell lines and primary samples (Yang et al., 2011).

ZKSCAN4, also known as ZNF307, encodes zinc finger with KRAB and SCANdomains 4 and is located on chromosome 6p21 (RefSeq, 2002). ZKSCAN4might suppress the p53-p21 pathway through activating MDM2 and EP300expression and inducing p53 degradation (Li et al., 2007).

ZMYM1 encodes zinc finger MYM-type containing 1 and is located onchromosome 1 p34.3 (RefSeq, 2002). ZMYM1 is a major interactor of ZNF131which acts in estrogen signaling and breast cancer proliferation (Oh andChung, 2012; Kim et al., 2016a).

DETAILED DESCRIPTION OF THE INVENTION

Stimulation of an immune response is dependent upon the presence ofantigens recognized as foreign by the host immune system. The discoveryof the existence of tumor associated antigens has raised the possibilityof using a host's immune system to intervene in tumor growth. Variousmechanisms of hamessing both the humoral and cellular arms of the immunesystem are currently being explored for cancer immunotherapy.

Specific elements of the cellular immune response are capable ofspecifically recognizing and destroying tumor cells. The isolation ofT-cells from tumor-infiltrating cell populations or from peripheralblood suggests that such cells play an important role in natural immunedefense against cancer. CD8-positive T-cells in particular, whichrecognize class I molecules of the major histocompatibility complex(MHC)-bearing peptides of usually 8 to 10, 8-11, 8-12 or 8-14 amino acidresidues derived from proteins or defect ribosomal products (DRIPS)located in the cytosol, play an important role in this response. TheMHC-molecules of the human are also designated as humanleukocyte-antigens (HLA).

The term “T-cell response” means the specific proliferation andactivation of effector functions induced by a peptide in vitro or invivo. For MHC class I restricted cytotoxic T cells, effector functionsmay be lysis of peptide-pulsed, peptide-precursor pulsed or naturallypeptide-presenting target cells, secretion of cytokines, preferablyInterferon-gamma, TNF-alpha, or IL-2 induced by peptide, secretion ofeffector molecules, preferably granzymes or perforins induced bypeptide, or degranulation.

The term “peptide” is used herein to designate a series of amino acidresidues, connected one to the other typically by peptide bonds betweenthe alpha-amino and carbonyl groups of the adjacent amino acids. Thepeptides are preferably 9 amino acids in length, but can be as short as8 amino acids in length, and as long as 10, 11, or 12 or longer, and incase of MHC class II peptides (elongated variants of the peptides of theinvention) they can be as long as 13, 14, 15, 16, 17, 18, 19 or 20 ormore amino acids in length.

Furthermore, the term “peptide” shall include salts of a series of aminoacid residues, connected one to the other typically by peptide bondsbetween the alpha-amino and carbonyl groups of the adjacent amino acids.Preferably, the salts are pharmaceutical acceptable salts of thepeptides, such as, for example, the chloride or acetate(trifluoroacetate) salts. It has to be noted that the salts of thepeptides according to the present invention differ substantially fromthe peptides in their state(s) in vivo, as the peptides are not salts invivo.

The term “peptide” shall also include “oligopeptide”. The term“oligopeptide” is used herein to designate a series of amino acidresidues, connected one to the other typically by peptide bonds betweenthe alpha-amino and carbonyl groups of the adjacent amino acids. Thelength of the oligopeptide is not critical to the invention, as long asthe correct epitope or epitopes are maintained therein. Theoligopeptides are typically less than about 30 amino acid residues inlength, and greater than about 15 amino acids in length.

The term “polypeptide” designates a series of amino acid residues,connected one to the other typically by peptide bonds between thealpha-amino and carbonyl groups of the adjacent amino acids. The lengthof the polypeptide is not critical to the invention as long as thecorrect epitopes are maintained. In contrast to the terms peptide oroligopeptide, the term polypeptide is meant to refer to moleculescontaining more than about 30 amino acid residues.

A peptide, oligopeptide, protein or polynucleotide coding for such amolecule is “immunogenic” (and thus is an “immunogen” within the presentinvention), if it is capable of inducing an immune response. In the caseof the present invention, immunogenicity is more specifically defined asthe ability to induce a T-cell response. Thus, an “immunogen” would be amolecule that is capable of inducing an immune response, and in the caseof the present invention, a molecule capable of inducing a T-cellresponse. In another aspect, the immunogen can be the peptide, thecomplex of the peptide with MHC, oligopeptide, and/or protein that isused to raise specific antibodies or TCRs against it.

A class I T cell “epitope” requires a short peptide that is bound to aclass I MHC receptor, forming a ternary complex (MHC class I alphachain, beta-2-microglobulin, and peptide) that can be recognized by a Tcell bearing a matching T-cell receptor binding to the MHC/peptidecomplex with appropriate affinity. Peptides binding to MHC class Imolecules are typically 8-10, 8-11, 8-12 or 8-14 amino acids in length,and most typically 9 amino acids in length.

In humans, there are three different genetic loci that encode MHC classI molecules (the MHC-molecules of the human are also designated humanleukocyte antigens (HLA)): HLA-A, HLA-B, and HLA-C. HLA-A*01, HLA-A*02,and HLA-B*07 are examples of different MHC class I alleles that can beexpressed from these loci.

TABLE 5 Expression frequencies (F) of HLA-A*02 and HLA-A*24 and the mostfrequent HLA-DR serotypes. Frequencies are deduced from haplotypefrequencies Gf within the American population adapted from Mori et al.(Mori et al., 1997) employing the Hardy-Weinberg formula F = 1 − (1 −Gf)². Combinations of A*02 or A*24 with certain HLA-DR alleles might beenriched or less frequent than expected from their single frequenciesdue to linkage disequilibrium. For details refer to Chanock et al.(Chanock et al., 2004). Calculated phenotype Allele Population fromallele frequency A*02 Caucasian (North America)  49.1% A*02 AfricanAmerican (North America)  34.1% A*02 Asian American (North America) 43.2% A*02 Latin American (North American)  48.3% DR1 Caucasian (NorthAmerica)  19.4% DR2 Caucasian (North America)  28.2% DR3 Caucasian(North America)  20.6% DR4 Caucasian (North America)  30.7% DR5Caucasian (North America)  23.3% DR6 Caucasian (North America)  26.7%DR7 Caucasian (North America)  24.8% DR8 Caucasian (North America)  5.7%DR9 Caucasian (North America)  2.1% DR1 African (North) American 13.20%DR2 African (North) American 29.80% DR3 African (North) American 24.80%DR4 African (North) American 11.10% DR5 African (North) American 31.10%DR6 African (North) American 33.70% DR7 African (North) American 19.20%DR8 African (North) American 12.10% DR9 African (North) American  5.80%DR1 Asian (North) American  6.80% DR2 Asian (North) American 33.80% DR3Asian (North) American  9.20% DR4 Asian (North) American 28.60% DR5Asian (North) American 30.00% DR6 Asian (North) American 25.10% DR7Asian (North) American 13.40% DR8 Asian (North) American 12.70% DR9Asian (North) American 18.60% DR1 Latin (North) American 15.30% DR2Latin (North) American 21.20% DR3 Latin (North) American 15.20% DR4Latin (North) American 36.80% DR5 Latin (North) American 20.00% DR6Latin (North) American 31.10% DR7 Latin (North) American 20.20% DR8Latin (North) American 18.60% DR9 Latin (North) American  2.10% A*24Philippines   65% A*24 Russia Nenets   61% A*24:02 Japan   59% A*24Malaysia   58% A*24:02 Philippines   54% A*24 India   47% A*24 SouthKorea   40% A*24 Sri Lanka   37% A*24 China   32% A*24:02 India   29%A*24 Australia West   22% A*24 USA   22% A*24 Russia Samara   20% A*24South America   20% A*24 Europe   18%

The peptides of the invention, preferably when included into a vaccineof the invention as described herein bind to A*02. A vaccine may alsoinclude pan-binding MHC class II peptides. Therefore, the vaccine of theinvention can be used to treat cancer in patients that are A*02positive, whereas no selection for MHC class II allotypes is necessarydue to the pan-binding nature of these peptides.

If A*02 peptides of the invention are combined with peptides binding toanother allele, for example A*24, a higher percentage of any patientpopulation can be treated compared with addressing either MHC class Iallele alone. While in most populations less than 50% of patients couldbe addressed by either allele alone, a vaccine comprising HLA-A*24 andHLA-A*02 epitopes can treat at least 60% of patients in any relevantpopulation. Specifically, the following percentages of patients will bepositive for at least one of these alleles in various regions: USA 61%,Western Europe 62%, China 75%, South Korea 77%, Japan 86% (calculatedfrom www.allelefrequencies.net).

In a preferred embodiment, the term “nucleotide sequence” refers to aheteropolymer of deoxyribonucleotides.

The nucleotide sequence coding for a particular peptide, oligopeptide,or polypeptide may be naturally occurring or they may be syntheticallyconstructed. Generally, DNA segments encoding the peptides,polypeptides, and proteins of this invention are assembled from cDNAfragments and short oligonucleotide linkers, or from a series ofoligonucleotides, to provide a synthetic gene that is capable of beingexpressed in a recombinant transcriptional unit comprising regulatoryelements derived from a microbial or viral operon.

As used herein the term “a nucleotide coding for (or encoding) apeptide” refers to a nucleotide sequence coding for the peptideincluding artificial (man-made) start and stop codons compatible for thebiological system the sequence is to be expressed by, for example, adendritic cell or another cell system useful for the production of TCRs.

As used herein, reference to a nucleic acid sequence includes bothsingle stranded and double stranded nucleic acid. Thus, for example forDNA, the specific sequence, unless the context indicates otherwise,refers to the single strand DNA of such sequence, the duplex of suchsequence with its complement (double stranded DNA) and the complement ofsuch sequence.

The term “coding region” refers to that portion of a gene which eithernaturally or normally codes for the expression product of that gene inits natural genomic environment, i.e., the region coding in vivo for thenative expression product of the gene.

The coding region can be derived from a non-mutated (“normal”), mutatedor altered gene, or can even be derived from a DNA sequence, or gene,wholly synthesized in the laboratory using methods well known to thoseof skill in the art of DNA synthesis.

The term “expression product” means the polypeptide or protein that isthe natural translation product of the gene and any nucleic acidsequence coding equivalents resulting from genetic code degeneracy andthus coding for the same amino acid(s).

The term “fragment”, when referring to a coding sequence, means aportion of DNA comprising less than the complete coding region, whoseexpression product retains essentially the same biological function oractivity as the expression product of the complete coding region.

The term “DNA segment” refers to a DNA polymer, in the form of aseparate fragment or as a component of a larger DNA construct, which hasbeen derived from DNA isolated at least once in substantially pure form,i.e., free of contaminating endogenous materials and in a quantity orconcentration enabling identification, manipulation, and recovery of thesegment and its component nucleotide sequences by standard biochemicalmethods, for example, by using a cloning vector. Such segments areprovided in the form of an open reading frame uninterrupted by internalnon-translated sequences, or introns, which are typically present ineukaryotic genes. Sequences of non-translated DNA may be presentdownstream from the open reading frame, where the same do not interferewith manipulation or expression of the coding regions.

The term “primer” means a short nucleic acid sequence that can be pairedwith one strand of DNA and provides a free 3′-OH end at which a DNApolymerase starts synthesis of a deoxyribonucleotide chain.

The term “promoter” means a region of DNA involved in binding of RNApolymerase to initiate transcription.

The term “isolated” means that the material is removed from its originalenvironment (e.g., the natural environment, if it is naturallyoccurring). For example, a naturally-occurring polynucleotide orpolypeptide present in a living animal is not isolated, but the samepolynucleotide or polypeptide, separated from some or all of thecoexisting materials in the natural system, is isolated. Suchpolynucleotides could be part of a vector and/or such polynucleotides orpolypeptides could be part of a composition, and still be isolated inthat such vector or composition is not part of its natural environment.

The polynucleotides, and recombinant or immunogenic polypeptides,disclosed in accordance with the present invention may also be in“purified” form. The term “purified” does not require absolute purity;rather, it is intended as a relative definition, and can includepreparations that are highly purified or preparations that are onlypartially purified, as those terms are understood by those of skill inthe relevant art. For example, individual clones isolated from a cDNAlibrary have been conventionally purified to electrophoretichomogeneity. Purification of starting material or natural material to atleast one order of magnitude, preferably two or three orders, and morepreferably four or five orders of magnitude is expressly contemplated.Furthermore, a claimed polypeptide which has a purity of preferably99.999%, or at least 99.99% or 99.9%; and even desirably 99% by weightor greater is expressly encompassed.

The nucleic acids and polypeptide expression products disclosedaccording to the present invention, as well as expression vectorscontaining such nucleic acids and/or such polypeptides, may be in“enriched form”. As used herein, the term “enriched” means that theconcentration of the material is at least about 2, 5, 10, 100, or 1000times its natural concentration (for example), advantageously 0.01%, byweight, preferably at least about 0.1% by weight. Enriched preparationsof about 0.5%, 1%, 5%, 10%, and 20% by weight are also contemplated. Thesequences, constructs, vectors, clones, and other materials comprisingthe present invention can advantageously be in enriched or isolatedform. The term “active fragment” means a fragment, usually of a peptide,polypeptide or nucleic acid sequence, that generates an immune response(i.e., has immunogenic activity) when administered, alone or optionallywith a suitable adjuvant or in a vector, to an animal, such as a mammal,for example, a rabbit or a mouse, and also including a human, suchimmune response taking the form of stimulating a T-cell response withinthe recipient animal, such as a human. Alternatively, the “activefragment” may also be used to induce a T-cell response in vitro.

As used herein, the terms “portion”, “segment” and “fragment”, when usedin relation to polypeptides, refer to a continuous sequence of residues,such as amino acid residues, which sequence forms a subset of a largersequence. For example, if a polypeptide were subjected to treatment withany of the common endopeptidases, such as trypsin or chymotrypsin, theoligopeptides resulting from such treatment would represent portions,segments or fragments of the starting polypeptide. When used in relationto polynucleotides, these terms refer to the products produced bytreatment of said polynucleotides with any of the endonucleases.

In accordance with the present invention, the term “percent identity” or“percent identical”, when referring to a sequence, means that a sequenceis compared to a claimed or described sequence after alignment of thesequence to be compared (the “Compared Sequence”) with the described orclaimed sequence (the “Reference Sequence”). The percent identity isthen determined according to the following formula:

percent identity=100[1−(C/R)]

wherein C is the number of differences between the Reference Sequenceand the Compared Sequence over the length of alignment between theReference Sequence and the Compared Sequence, wherein(i) each base or amino acid in the Reference Sequence that does not havea corresponding aligned base or amino acid in the Compared Sequence and(ii) each gap in the Reference Sequence and(iii) each aligned base or amino acid in the Reference Sequence that isdifferent from an aligned base or amino acid in the Compared Sequence,constitutes a difference and(iv) the alignment has to start at position 1 of the aligned sequences;and R is the number of bases or amino acids in the Reference Sequenceover the length of the alignment with the Compared Sequence with any gapcreated in the Reference Sequence also being counted as a base or aminoacid.

If an alignment exists between the Compared Sequence and the ReferenceSequence for which the percent identity as calculated above is aboutequal to or greater than a specified minimum Percent Identity then theCompared Sequence has the specified minimum percent identity to theReference Sequence even though alignments may exist in which the hereinabove calculated percent identity is less than the specified percentidentity.

As mentioned above, the present invention thus provides a peptidecomprising a sequence that is selected from the group of consisting ofSEQ ID NO: 1 to SEQ ID NO: 32 or a variant thereof which is 88%homologous to SEQ ID NO: 1 to SEQ ID NO: 32, or a variant thereof thatwill induce T cells cross-reacting with said peptide. The peptides ofthe invention have the ability to bind to a molecule of the human majorhistocompatibility complex (MHC) class-I or elongated versions of saidpeptides to class II.

In the present invention, the term “homologous” refers to the degree ofidentity (see percent identity above) between sequences of two aminoacid sequences, i.e. peptide or polypeptide sequences. Theaforementioned “homology” is determined by comparing two sequencesaligned under optimal conditions over the sequences to be compared. Sucha sequence homology can be calculated by creating an alignment using,for example, the ClustalW algorithm. Commonly available sequenceanalysis software, more specifically, Vector NTI, GENETYX or other toolsare provided by public databases.

A person skilled in the art will be able to assess, whether T cellsinduced by a variant of a specific peptide will be able to cross-reactwith the peptide itself (Appay et al., 2006; Colombetti et al., 2006;Fong et al., 2001; Zaremba et al., 1997).

By a “variant” of the given amino acid sequence the inventors mean thatthe side chains of, for example, one or two of the amino acid residuesare altered (for example by replacing them with the side chain ofanother naturally occurring amino acid residue or some other side chain)such that the peptide is still able to bind to an HLA molecule insubstantially the same way as a peptide consisting of the given aminoacid sequence in consisting of SEQ ID NO: 1 to SEQ ID NO: 32. Forexample, a peptide may be modified so that it at least maintains, if notimproves, the ability to interact with and bind to the binding groove ofa suitable MHC molecule, such as HLA-A*02 or -DR, and in that way it atleast maintains, if not improves, the ability to bind to the TCR ofactivated T cells.

These T cells can subsequently cross-react with cells and kill cellsthat express a polypeptide that contains the natural amino acid sequenceof the cognate peptide as defined in the aspects of the invention. Ascan be derived from the scientific literature and databases (Rammenseeet al., 1999; Godkin et al., 1997), certain positions of HLA bindingpeptides are typically anchor residues forming a core sequence fittingto the binding motif of the HLA receptor, which is defined by polar,electrophysical, hydrophobic and spatial properties of the polypeptidechains constituting the binding groove. Thus, one skilled in the artwould be able to modify the amino acid sequences set forth in SEQ ID NO:1 to SEQ ID NO 32, by maintaining the known anchor residues, and wouldbe able to determine whether such variants maintain the ability to bindMHC class I or II molecules. The variants of the present inventionretain the ability to bind to the TCR of activated T cells, which cansubsequently cross-react with and kill cells that express a polypeptidecontaining the natural amino acid sequence of the cognate peptide asdefined in the aspects of the invention.

The original (unmodified) peptides as disclosed herein can be modifiedby the substitution of one or more residues at different, possiblyselective, sites within the peptide chain, if not otherwise stated.Preferably those substitutions are located at the end of the amino acidchain. Such substitutions may be of a conservative nature, for example,where one amino acid is replaced by an amino acid of similar structureand characteristics, such as where a hydrophobic amino acid is replacedby another hydrophobic amino acid. Even more conservative would bereplacement of amino acids of the same or similar size and chemicalnature, such as where leucine is replaced by isoleucine. In studies ofsequence variations in families of naturally occurring homologousproteins, certain amino acid substitutions are more often tolerated thanothers, and these are often show correlation with similarities in size,charge, polarity, and hydrophobicity between the original amino acid andits replacement, and such is the basis for defining “conservativesubstitutions.” Conservative substitutions are herein defined asexchanges within one of the following five groups: Group 1-smallaliphatic, nonpolar or slightly polar residues (Ala, Ser, Thr, Pro,Gly); Group 2-polar, negatively charged residues and their amides (Asp,Asn, Glu, Gin); Group 3-polar, positively charged residues (His, Arg,Lys); Group 4-large, aliphatic, nonpolar residues (Met, Leu, lie, Val,Cys); and Group 5-large, aromatic residues (Phe, Tyr, Trp).

Less conservative substitutions might involve the replacement of oneamino acid by another that has similar characteristics but is somewhatdifferent in size, such as replacement of an alanine by an isoleucineresidue. Highly non-conservative replacements might involve substitutingan acidic amino acid for one that is polar, or even for one that isbasic in character. Such “radical” substitutions cannot, however, bedismissed as potentially ineffective since chemical effects are nottotally predictable and radical substitutions might well give rise toserendipitous effects not otherwise predictable from simple chemicalprinciples.

Of course, such substitutions may involve structures other than thecommon L-amino acids. Thus, D-amino acids might be substituted for theL-amino acids commonly found in the antigenic peptides of the inventionand yet still be encompassed by the disclosure herein. In addition,non-standard amino acids (i.e., other than the common naturallyoccurring proteinogenic amino acids) may also be used for substitutionpurposes to produce immunogens and immunogenic polypeptides according tothe present invention.

If substitutions at more than one position are found to result in apeptide with substantially equivalent or greater antigenic activity asdefined below, then combinations of those substitutions will be testedto determine if the combined substitutions result in additive orsynergistic effects on the antigenicity of the peptide. At most, no morethan 4 positions within the peptide would be simultaneously substituted.

A peptide consisting essentially of the amino acid sequence as indicatedherein can have one or two non-anchor amino acids (see below regardingthe anchor motif) exchanged without that the ability to bind to amolecule of the human major histocompatibility complex (MHC) class-I or—II is substantially changed or is negatively affected, when compared tothe non-modified peptide. In another embodiment, in a peptide consistingessentially of the amino acid sequence as indicated herein, one or twoamino acids can be exchanged with their conservative exchange partners(see herein below) without that the ability to bind to a molecule of thehuman major histocompatibility complex (MHC) class-I or —II issubstantially changed, or is negatively affected, when compared to thenon-modified peptide.

The amino acid residues that do not substantially contribute tointeractions with the T-cell receptor can be modified by replacementwith other amino acid whose incorporation does not substantially affectT-cell reactivity and does not eliminate binding to the relevant MHC.Thus, apart from the proviso given, the peptide of the invention may beany peptide (by which term the inventors include oligopeptide orpolypeptide), which includes the amino acid sequences or a portion orvariant thereof as given.

TABLE 6 Variants and motif of the peptides according toSEQ ID NO: 4, 5, and 7 Position 1 2 3 4 5 6 7 8 9 10 11 SEQ ID NO. 4 F LL E A Q N Y L Variants V I A M V M I M M A A V A I A A A V V V I V V A TV T I T T A Q V Q I Q Q A SEQ ID NO. 5 G L I D E V M V L L V I A M V M IM M A A V A I A A A V V V I V V A T V T I T T A Q V Q I Q Q ASEQ ID NO. 7 L L L S D P D K V T I Variants V L A M V M M L M A A V A AL A A V V V V L V A T V T T L T A Q V Q Q L Q A

Longer (elongated) peptides may also be suitable. It is possible thatMHC class I epitopes, although usually between 8 and 0, 8-11, 8-12 or8-14 amino acids long, are generated by peptide processing from longerpeptides or proteins that include the actual epitope. It is preferredthat the residues that flank the actual epitope are residues that do notsubstantially affect proteolytic cleavage necessary to expose the actualepitope during processing.

The peptides of the invention can be elongated by up to four aminoacids, that is 1, 2, 3 or 4 amino acids can be added to either end inany combination between 4:0 and 0:4. Combinations of the elongationsaccording to the invention can be found in Table 7.

TABLE 7 Combinations of the elongations of peptides of the inventionC-terminus N-terminus 4 0 3 0 or 1 2 0 or 1 or 2 C-terminus N-terminus 10 or 1 or 2 or 3 0 0 or 1 or 2 or 3 or 4 N-terminus C-terminus 4 0 3 0or 1 2 0 or 1 or 2 1 0 or 1 or 2 or 3 0 0 or 1 or 2 or 3 or 4

The amino acids for the elongation/extension can be the peptides of theoriginal sequence of the protein or any other amino acid(s). Theelongation can be used to enhance the stability or solubility of thepeptides.

Thus, the epitopes of the present invention may be identical tonaturally occurring tumor-associated or tumor-specific epitopes or mayinclude epitopes that differ by no more than four residues from thereference peptide, as long as they have substantially identicalantigenic activity.

In an alternative embodiment, the peptide is elongated on either or bothsides by more than 4 amino acids, preferably to a total length of up to30 amino acids. This may lead to MHC class II binding peptides. Bindingto MHC class II can be tested by methods known in the art.

Accordingly, the present invention provides peptides and variants of MHCclass I epitopes, wherein the peptide or variant has an overall lengthof from 8 and 100, from 9 and 100, from 10 and 100, from 11 and 100,from 12 and 100, preferably from 8 and 30, and from 9 and 30, from 10and 30, from 11 and 30, from 12 and 30, most preferred from 8 and 14,from 9 and 14, from 10 and 14, from 11 and 14, from 12 and 14. Thepresent invention further provides peptides and variants of MHC class Iepitopes, wherein the peptide or variant has an overall length of namely8, 9, 10, 11, 12, 13, or 14 amino acids, in case of the elongated classII binding peptides the length can also be 15, 16, 17, 18, 19, 20, 21 or22 amino acids.

Of course, the peptide or variant according to the present inventionwill have the ability to bind to a molecule of the human majorhistocompatibility complex (MHC) class I or II.

Binding of a peptide or a variant to a MHC complex may be tested bymethods known in the art.

Preferably, when the T cells specific for a peptide according to thepresent invention are tested against the substituted peptides, thepeptide concentration at which the substituted peptides achieve half themaximal increase in lysis relative to background is no more than about 1mM, preferably no more than about 1 μM, more preferably no more thanabout 1 nM, and still more preferably no more than about 100 μM, andmost preferably no more than about 10 μM. It is also preferred that thesubstituted peptide be recognized by T cells from more than oneindividual, at least two, and more preferably three individuals.

In a particularly preferred embodiment of the invention the peptideconsists of or consists essentially of an amino acid sequence accordingto SEQ ID NO: 1 to SEQ ID NO: 32.

“Consisting essentially of” shall mean that a peptide according to thepresent invention, in addition to the sequence according to any of SEQID NO: 1 to SEQ ID NO 32 or a variant thereof contains additional N-and/or C-terminally located stretches of amino acids that are notnecessarily forming part of the peptide that functions as an epitope forMHC molecules epitope.

Nevertheless, these stretches can be important to provide an efficientintroduction of the peptide according to the present invention into thecells. In one embodiment of the present invention, the peptide is partof a fusion protein which comprises, for example, the 80 N-terminalamino acids of the HLA-DR antigen-associated invariant chain (p33, inthe following “Ii”) as derived from the NCBI, GenBank Accession numberX00497. In other fusions, the peptides of the present invention can befused to an antibody as described herein, or a functional part thereof,in particular into a sequence of an antibody, so as to be specificallytargeted by said antibody, or, for example, to or into an antibody thatis specific for proteins presented by dendritic cells as describedherein.

In addition, the peptide or variant may be modified further to improvestability and/or binding to MHC molecules in order to elicit a strongerimmune response. Methods for such an optimization of a peptide sequenceare well known in the art and include, for example, the introduction ofreverse peptide bonds or non-peptide bonds.

In a reverse peptide bond amino acid residues are not joined by peptide(—CO—NH—) linkages but the peptide bond is reversed. Such retro-inversopeptidomimetics may be made using methods known in the art, for examplesuch as those described in Meziere et al (1997) (Meziere et al., 1997),incorporated herein by reference. This approach involves makingpseudopeptides containing changes involving the backbone, and not theorientation of side chains. Meziere et al. (Meziere et al., 1997) showthat for MHC binding and T helper cell responses, these pseudopeptidesare useful. Retro-inverse peptides, which contain NH—CO bonds instead ofCO—NH peptide bonds, are much more resistant to proteolysis.

A non-peptide bond is, for example, —CH₂—NH, —CH₂S—, —CH₂CH₂—, —CH═CH—,—COCH₂—, —CH(OH)CH₂—, and —CH₂SO—. U.S. Pat. No. 4,897,445 provides amethod for the solid phase synthesis of non-peptide bonds (—CH₂—NH) inpolypeptide chains which involves polypeptides synthesized by standardprocedures and the non-peptide bond synthesized by reacting an aminoaldehyde and an amino acid in the presence of NaCNBH₃.

Peptides comprising the sequences described above may be synthesizedwith additional chemical groups present at their amino and/or carboxytermini, to enhance the stability, bioavailability, and/or affinity ofthe peptides. For example, hydrophobic groups such as carbobenzoxyl,dansyl, or t-butyloxycarbonyl groups may be added to the peptides' aminotermini. Likewise, an acetyl group or a 9-fluorenylmethoxy-carbonylgroup may be placed at the peptides' amino termini. Additionally, thehydrophobic group, t-butyloxycarbonyl, or an amido group may be added tothe peptides' carboxy termini.

Further, the peptides of the invention may be synthesized to alter theirsteric configuration. For example, the D-isomer of one or more of theamino acid residues of the peptide may be used, rather than the usualL-isomer. Still further, at least one of the amino acid residues of thepeptides of the invention may be substituted by one of the well-knownnon-naturally occurring amino acid residues. Alterations such as thesemay serve to increase the stability, bioavailability and/or bindingaction of the peptides of the invention.

Similarly, a peptide or variant of the invention may be modifiedchemically by reacting specific amino acids either before or aftersynthesis of the peptide. Examples for such modifications are well knownin the art and are summarized e.g. in R. Lundblad, Chemical Reagents forProtein Modification, 3rd ed. CRC Press, 2004 (Lundblad, 2004), which isincorporated herein by reference. Chemical modification of amino acidsincludes but is not limited to, modification by acylation, amidination,pyridoxylation of lysine, reductive alkylation, trinitrobenzylation ofamino groups with 2,4,6-trinitrobenzene sulphonic acid (TNBS), amidemodification of carboxyl groups and sulphydryl modification by performicacid oxidation of cysteine to cysteic acid, formation of mercurialderivatives, formation of mixed disulphides with other thiol compounds,reaction with maleimide, carboxymethylation with iodoacetic acid oriodoacetamide and carbamoylation with cyanate at alkaline pH, althoughwithout limitation thereto. In this regard, the skilled person isreferred to Chapter 15 of Current Protocols In Protein Science, Eds.Coligan et al. (John Wiley and Sons NY 1995-2000) (Coligan et al., 1995)for more extensive methodology relating to chemical modification ofproteins.

Briefly, modification of e.g. arginyl residues in proteins is oftenbased on the reaction of vicinal dicarbonyl compounds such asphenylglyoxal, 2,3-butanedione, and 1,2-cyclohexanedione to form anadduct. Another example is the reaction of methylglyoxal with arginineresidues. Cysteine can be modified without concomitant modification ofother nucleophilic sites such as lysine and histidine. As a result, alarge number of reagents are available for the modification of cysteine.The websites of companies such as Sigma-Aldrich (www.sigma-aldrich.com)provide information on specific reagents.

Selective reduction of disulfide bonds in proteins is also common.Disulfide bonds can be formed and oxidized during the heat treatment ofbiopharmaceuticals. Woodward's Reagent K may be used to modify specificglutamic acid residues. N-(3-(dimethylamino)propyl)-N′-ethylcarbodiimidecan be used to form intra-molecular crosslinks between a lysine residueand a glutamic acid residue. For example, diethylpyrocarbonate is areagent for the modification of histidyl residues in proteins.

Histidine can also be modified using 4-hydroxy-2-nonenal. The reactionof lysine residues and other α-amino groups is, for example, useful inbinding of peptides to surfaces or the cross-linking ofproteins/peptides. Lysine is the site of attachment ofpoly(ethylene)glycol and the major site of modification in theglycosylation of proteins. Methionine residues in proteins can bemodified with e.g. iodoacetamide, bromoethylamine, and chloramine T.

Tetranitromethane and N-acetylimidazole can be used for the modificationof tyrosyl residues. Cross-linking via the formation of dityrosine canbe accomplished with hydrogen peroxide/copper ions.

Recent studies on the modification of tryptophan have usedN-bromosuccinimide, 2-hydroxy-5-nitrobenzyl bromide or3-bromo-3-methyl-2-(2-nitrophenylmercapto)-3H-indole (BPNS-skatole).

Successful modification of therapeutic proteins and peptides with PEG isoften associated with an extension of circulatory half-life whilecross-linking of proteins with glutaraldehyde, polyethylene glycoldiacrylate and formaldehyde is used for the preparation of hydrogels.

Chemical modification of allergens for immunotherapy is often achievedby carbamylation with potassium cyanate.

A peptide or variant, wherein the peptide is modified or includesnon-peptide bonds is a preferred embodiment of the invention. Anotherembodiment of the present invention relates to a non-naturally occurringpeptide wherein said peptide consists or consists essentially of anamino acid sequence according to SEQ ID No: 1 to SEQ ID No: 32 and hasbeen synthetically produced (e.g. synthesized) as a pharmaceuticallyacceptable salt.

Methods to synthetically produce peptides are well known in the art. Thesalts of the peptides according to the present invention differsubstantially from the peptides in their state(s) in vivo, as thepeptides as generated in vivo are no salts. The non-natural salt form ofthe peptide mediates the solubility of the peptide, in particular in thecontext of pharmaceutical compositions comprising the peptides, e.g. thepeptide vaccines as disclosed herein. A sufficient and at leastsubstantial solubility of the peptide(s) is required in order toefficiently provide the peptides to the subject to be treated.Preferably, the salts are pharmaceutically acceptable salts of thepeptides. These salts according to the invention include alkaline andearth alkaline salts such as salts of the Hofmeister series comprisingas anions PO₄ ³⁻, SO₄ ²⁻, CH₃COO⁻, Cl⁻, Br⁻, NO₃ ⁻, ClO₄ ⁻, I⁻, SCN⁻ andas cations NH₄ ⁺, Rb⁺, K⁺, Na⁺, Cs⁺, Li⁺, Zn²⁺, Mg²⁺, Ca²⁺, Mn²⁺, Cu²⁺and Ba²⁺. Particularly salts are selected from (NH₄)₃PO₄, (NH₄)₂HPO₄,(NH₄)H₂PO₄, (NH₄)₂SO₄, NH₄CH₃COO, NH₄Cl, NH₄Br, NH₄NO₃, NH₄ClO₄, NH₄I,NH₄SCN, Rb₃PO₄, Rb₂HPO₄, RbH₂PO₄, Rb₂SO₄, Rb₄CH₃COO, Rb₄Cl, Rb₄Br,Rb₄NO₃, Rb₄ClO₄, Rb₄I, Rb₄SCN, K₃PO₄, K₂HPO₄, KH₂PO₄, K₂SO₄, KCH₃COO,KCl, KBr, KNO₃, KClO₄, KI, KSCN, Na₃PO₄, Na₂HPO₄, NaH₂PO₄, Na₂SO₄,NaCH₃COO, NaCl, NaBr, NaNO₃, NaClO₄, NaI, NaSCN, ZnCl₂ Cs₃PO₄, Cs₂HPO₄,CsH₂PO₄, Cs₂SO₄, CsCH₃COO, CsCl, CsBr, CsNO₃, CsClO₄, CsI, CsSCN,Li₃PO₄, Li₂HPO₄, LiH₂PO₄, Li₂SO₄, LiCH₃COO, LiCl, LiBr, LiNO₃, LiClO₄,LiI, LiSCN, Cu₂SO₄, Mg₃(PO₄)₂, Mg₂HPO₄, Mg(H₂PO₄)₂, Mg₂SO₄, Mg(CH₃COO)₂,MgCl₂, MgBr₂, Mg(NOs₃)₂, Mg(ClO₄)₂, MgI₂, Mg(SCN)₂, MnCl₂, Ca₃(PO₄),Ca₂HPO₄, Ca(H₂PO₄)₂, CaSO₄, Ca(CHCOO)₂, CaCl₂, CaBr₂, Ca(NOs₃)₂,Ca(ClO₄)₂, CaI₂, Ca(SCN)₂, Ba₃(PO₄)₂, Ba₂HPO₄, Ba(H₂PO₄)₂, BaSO₄,Ba(CHCOO)₂, BaCl₂, BaBr₂, Ba(NOs₃)₂, Ba(ClO₄)₂, BaI₂, and Ba(SCN)₂.Particularly preferred are NH acetate, MgCl₂, KH₂PO₄, Na₂SO₄, KCl, NaCl,and CaCl₂, such as, for example, the chloride or acetate(trifluoroacetate) salts.

Generally, peptides and variants (at least those containing peptidelinkages between amino acid residues) may be synthesized by theFmoc-polyamide mode of solid-phase peptide synthesis as disclosed byLukas et al. (Lukas et al., 1981) and by references as cited therein.Temporary N-amino group protection is afforded by the9-fluorenylmethyloxycarbonyl (Fmoc) group. Repetitive cleavage of thishighly base-labile protecting group is done using 20% piperidine in N,N-dimethytformamide. Side-chain functionalities may be protected astheir butyl ethers (in the case of serine threonine and tyrosine), butylesters (in the case of glutamic acid and aspartic acid),butyloxycarbonyl derivative (in the case of lysine and histidine),trityl derivative (in the case of cysteine) and4-methoxy-2,3,6-trimethylbenzenesulphonyl derivative (in the case ofarginine). Where glutamine or asparagine are C-terminal residues, use ismade of the 4,4′-dimethoxybenzhydryl group for protection of the sidechain amido functionalities. The solid-phase support is based on apolydimethyl-acrylamide polymer constituted from the three monomersdimethylacrylamide (backbone-monomer), bisacryloylethylene diamine(cross linker) and acryloylsarcosine methyl ester (functionalizingagent). The peptide-to-resin cleavable linked agent used is theacid-labile 4-hydroxymethyl-phenoxyacetic acid derivative. All aminoacid derivatives are added as their preformed symmetrical anhydridederivatives with the exception of asparagine and glutamine, which areadded using a reversed N, N-dicyclohexyl-carbodiimide/1hydroxybenzotriazole mediated coupling procedure. All coupling anddeprotection reactions are monitored using ninhydrin, trinitrobenzenesulphonic acid or isotin test procedures. Upon completion of synthesis,peptides are cleaved from the resin support with concomitant removal ofside-chain protecting groups by treatment with 95% trifluoracetic acidcontaining a 50% scavenger mix. Scavengers commonly used includeethanedithiol, phenol, anisole and water, the exact choice depending onthe constituent amino acids of the peptide being synthesized.

Also a combination of solid phase and solution phase methodologies forthe synthesis of peptides is possible (see, for example, (Bruckdorfer etal., 2004), and the references as cited therein).

Trifluoracetic acid is removed by evaporation in vacuo, with subsequenttrituration with diethyl ether affording the crude peptide. Anyscavengers present are removed by a simple extraction procedure which onlyophilization of the aqueous phase affords the crude peptide free ofscavengers. Reagents for peptide synthesis are generally available frome.g. Calbiochem-Novabiochem (Nottingham, UK).

Purification may be performed by any one, or a combination of,techniques such as re-crystallization, size exclusion chromatography,ion-exchange chromatography, hydrophobic interaction chromatography and(usually) reverse-phase high performance liquid chromatography usinge.g. acetonitrile/water gradient separation.

Analysis of peptides may be carried out using thin layer chromatography,electrophoresis, in particular capillary electrophoresis, solid phaseextraction (CSPE), reverse-phase high performance liquid chromatography,amino-acid analysis after acid hydrolysis and by fast atom bombardment(FAB) mass spectrometric analysis, as well as MALDI and ESI-Q-TOF massspectrometric analysis.

In order to select over-presented peptides, a presentation profile iscalculated showing the median sample presentation as well as replicatevariation. The profile juxtaposes samples of the tumor entity ofinterest to a baseline of normal tissue samples. Each of these profilescan then be consolidated into an over-presentation score by calculatingthe p-value of a Linear Mixed-Effects Model (Pinheiro et al., 2015)adjusting for multiple testing by False Discovery Rate (Benjamini andHochberg, 1995) (cf. Example 1, FIGS. 1A to A-1G).

For the identification and relative quantitation of HLA ligands by massspectrometry, HLA molecules from shock-frozen tissue samples werepurified and HLA-associated peptides were isolated. The isolatedpeptides were separated and sequences were identified by onlinenano-electrospray-ionization (nanoESI) liquid chromatography-massspectrometry (LC-MS) experiments. The resulting peptide sequences wereverified by comparison of the fragmentation pattern of naturaltumor-associated peptides (TUMAPs) recorded from gallbladder cancer andcholangiocarcinoma samples (N=17 A*02-positive samples) with thefragmentation patterns of corresponding synthetic reference peptides ofidentical sequences. Since the peptides were directly identified asligands of HLA molecules of primary tumors, these results provide directevidence for the natural processing and presentation of the identifiedpeptides on primary cancer tissue obtained from 17 gallbladder cancerand cholangiocarcinoma patients.

The discovery pipeline XPRESIDENT® v2.1 (see, for example, US2013-0096016, which is hereby incorporated by reference in its entirety)allows the identification and selection of relevant over-presentedpeptide vaccine candidates based on direct relative quantitation ofHLA-restricted peptide levels on cancer tissues in comparison to severaldifferent non-cancerous tissues and organs. This was achieved by thedevelopment of label-free differential quantitation using the acquiredLC-MS data processed by a proprietary data analysis pipeline, combiningalgorithms for sequence identification, spectral clustering, ioncounting, retention time alignment, charge state deconvolution andnormalization.

Presentation levels including error estimates for each peptide andsample were established. Peptides exclusively presented on tumor tissueand peptides over-presented in tumor versus non-cancerous tissues andorgans have been identified.

HLA-peptide complexes from gallbladder cancer and cholangiocarcinomatissue samples were purified and HLA-associated peptides were isolatedand analyzed by LC-MS (see examples). All TUMAPs contained in thepresent application were identified with this approach on primarygallbladder cancer and cholangiocarcinoma samples confirming theirpresentation on primary gallbladder cancer and cholangiocarcinoma.

TUMAPs identified on multiple gallbladder cancer and cholangiocarcinomaand normal tissues were quantified using ion-counting of label-freeLC-MS data. The method assumes that LC-MS signal areas of a peptidecorrelate with its abundance in the sample.

All quantitative signals of a peptide in various LC-MS experiments werenormalized based on central tendency, averaged per sample and mergedinto a bar plot, called presentation profile. The presentation profileconsolidates different analysis methods like protein database search,spectral clustering, charge state deconvolution (decharging) andretention time alignment and normalization.

Besides over-presentation of the peptide, mRNA expression of theunderlying gene was tested. mRNA data were obtained via RNASeq analysesof normal tissues and cancer tissues (cf. Example 2, FIGS. 2A to A-2C)).An additional source of normal tissue data was a database of publiclyavailable RNA expression data from around 3000 normal tissue samples(Lonsdale, 2013). Peptides which are derived from proteins whose codingmRNA is highly expressed in cancer tissue, but very low or absent invital normal tissues, were preferably included in the present invention.

The present invention provides peptides that are useful in treatingcancers/tumors, preferably gallbladder cancer and cholangiocarcinomathat over- or exclusively present the peptides of the invention. Thesepeptides were shown by mass spectrometry to be naturally presented byHLA molecules on primary human gallbladder cancer and cholangiocarcinomasamples.

Many of the source gene/proteins (also designated “full-length proteins”or “underlying proteins”) from which the peptides are derived were shownto be highly over-expressed in cancer compared with normaltissues—“normal tissues” in relation to this invention shall mean eitherhealthy gallbladder or bile duct cells or other normal tissue cells,demonstrating a high degree of tumor association of the source genes(see Example 2).

Moreover, the peptides themselves are strongly over-presented on tumortissue—“tumor tissue” in relation to this invention shall mean a samplefrom a patient suffering from gallbladder cancer and cholangiocarcinoma,but not on normal tissues (see Example 1).

HLA-bound peptides can be recognized by the immune system, specificallyT lymphocytes. T cells can destroy the cells presenting the recognizedHLA/peptide complex, e.g. gallbladder cancer and cholangiocarcinomacells presenting the derived peptides.

The peptides of the present invention have been shown to be capable ofstimulating T cell responses and/or are over-presented and thus can beused for the production of antibodies and/or TCRs, such as soluble TCRs,according to the present invention (see Example 3, Example 4).Furthermore, the peptides when complexed with the respective MHC can beused for the production of antibodies and/or TCRs, in particular sTCRs,according to the present invention, as well. Respective methods are wellknown to the person of skill, and can be found in the respectiveliterature as well. Thus, the peptides of the present invention areuseful for generating an immune response in a patient by which tumorcells can be destroyed. An immune response in a patient can be inducedby direct administration of the described peptides or suitable precursorsubstances (e.g. elongated peptides, proteins, or nucleic acids encodingthese peptides) to the patient, ideally in combination with an agentenhancing the immunogenicity (i.e. an adjuvant). The immune responseoriginating from such a therapeutic vaccination can be expected to behighly specific against tumor cells because the target peptides of thepresent invention are not presented on normal tissues in comparable copynumbers, preventing the risk of undesired autoimmune reactions againstnormal cells in the patient.

The present description further relates to T-cell receptors (TCRs)comprising an alpha chain and a beta chain (“alpha/beta TCRs”). Alsoprovided are peptides capable of binding to TCRs and antibodies whenpresented by an MHC molecule. The present description also relates tonucleic acids, vectors and host cells for expressing TCRs and peptidesof the present description; and methods of using the same.

The term “T-cell receptor” (abbreviated TCR) refers to a heterodimericmolecule comprising an alpha polypeptide chain (alpha chain) and a betapolypeptide chain (beta chain), wherein the heterodimeric receptor iscapable of binding to a peptide antigen presented by an HLA molecule.The term also includes so-called gamma/delta TCRs.

In one embodiment, the description provides a method of producing a TCRas described herein, the method comprising culturing a host cell capableof expressing the TCR under conditions suitable to promote expression ofthe TCR.

The description in another aspect relates to methods according to thedescription, wherein the antigen is loaded onto class I or II MHCmolecules expressed on the surface of a suitable antigen-presenting cellor artificial antigen-presenting cell by contacting a sufficient amountof the antigen with an antigen-presenting cell or the antigen is loadedonto class I or II MHC tetramers by tetramerizing the antigen/class I orII MHC complex monomers.

The alpha and beta chains of alpha/beta TCR's, and the gamma and deltachains of gamma/delta TCRs, are generally regarded as each having two“domains”, namely variable and constant domains. The variable domainconsists of a concatenation of variable region (V), and joining region(J). The variable domain may also include a leader region (L). Beta anddelta chains may also include a diversity region (D). The alpha and betaconstant domains may also include C-terminal transmembrane (TM) domainsthat anchor the alpha and beta chains to the cell membrane.

With respect to gamma/delta TCRs, the term “TCR gamma variable domain”as used herein refers to the concatenation of the TCR gamma V (TRGV)region without leader region (L), and the TCR gamma J (TRGJ) region, andthe term TCR gamma constant domain refers to the extracellular TRGCregion, or to a C-terminal truncated TRGC sequence. Likewise the term“TCR delta variable domain” refers to the concatenation of the TCR deltaV (TRDV) region without leader region (L) and the TCR delta D/J(TRDD/TRDJ) region, and the term “TCR delta constant domain” refers tothe extracellular TRDC region, or to a C-terminal truncated TRDCsequence.

TCRs of the present description preferably bind to a peptide-HLAmolecule complex with a binding affinity (KD) of about 100 μM or less,about 50 μM or less, about 25 μM or less, or about 10 μM or less. Morepreferred are high affinity TCRs having binding affinities of about 1 μMor less, about 100 nM or less, about 50 nM or less, about 25 nM or less.Non-limiting examples of preferred binding affinity ranges for TCRs ofthe present invention include about 1 nM to about 10 nM; about 10 nM toabout 20 nM; about 20 nM to about 30 nM; about 30 nM to about 40 nM;about 40 nM to about 50 nM; about 50 nM to about 60 nM; about 60 nM toabout 70 nM; about 70 nM to about 80 nM; about 80 nM to about 90 nM; andabout 90 nM to about 100 nM.

As used herein in connect with TCRs of the present description,“specific binding” and grammatical variants thereof are used to mean aTCR having a binding affinity (KD) for a peptide-HLA molecule complex of100 μM or less.

Alpha/beta heterodimeric TCRs of the present description may have anintroduced disulfide bond between their constant domains. Preferred TCRsof this type include those which have a TRAC constant domain sequenceand a TRBC1 or TRBC2 constant domain sequence except that Thr 48 of TRACand Ser 57 of TRBC1 or TRBC2 are replaced by cysteine residues, the saidcysteines forming a disulfide bond between the TRAC constant domainsequence and the TRBC1 or TRBC2 constant domain sequence of the TCR.

With or without the introduced inter-chain bond mentioned above,alpha/beta hetero-dimeric TCRs of the present description may have aTRAC constant domain sequence and a TRBC1 or TRBC2 constant domainsequence, and the TRAC constant domain sequence and the TRBC1 or TRBC2constant domain sequence of the TCR may be linked by the nativedisulfide bond between Cys4 of exon 2 of TRAC and Cys2 of exon 2 ofTRBC1 or TRBC2.

TCRs of the present description may comprise a detectable label selectedfrom the group consisting of a radionuclide, a fluorophore and biotin.TCRs of the present description may be conjugated to a therapeuticallyactive agent, such as a radionuclide, a chemotherapeutic agent, or atoxin.

In an embodiment, a TCR of the present description having at least onemutation in the alpha chain and/or having at least one mutation in thebeta chain has modified glycosylation compared to the unmutated TCR.

In an embodiment, a TCR comprising at least one mutation in the TCRalpha chain and/or TCR beta chain has a binding affinity for, and/or abinding half-life for, a peptide-HLA molecule complex, which is at leastdouble that of a TCR comprising the unmutated TCR alpha chain and/orunmutated TCR beta chain. Affinity-enhancement of tumor-specific TCRs,and its exploitation, relies on the existence of a window for optimalTCR affinities.

The existence of such a window is based on observations that TCRsspecific for HLA-A2-restricted pathogens have KD values that aregenerally about 10-fold lower when compared to TCRs specific forHLA-A2-restricted tumor-associated self-antigens. It is now known,although tumor antigens have the potential to be immunogenic, becausetumors arise from the individual's own cells only mutated proteins orproteins with altered translational processing will be seen as foreignby the immune system. Antigens that are upregulated or overexpressed (socalled self-antigens) will not necessarily induce a functional immuneresponse against the tumor: T-cells expressing TCRs that are highlyreactive to these antigens will have been negatively selected within thethymus in a process known as central tolerance, meaning that onlyT-cells with low-affinity TCRs for self-antigens remain. Therefore,affinity of TCRs or variants of the present description to the peptidesaccording to the invention can be enhanced by methods well known in theart.

The present description further relates to a method of identifying andisolating a TCR according to the present description, said methodcomprising incubating PBMCs from HLA-A*02-negative healthy donors withA2/peptide monomers, incubating the PBMCs with tetramer-phycoerythrin(PE) and isolating the high avidity T-cells by fluorescence activatedcell sorting (FACS)-Calibur analysis.

The present description further relates to a method of identifying andisolating a TCR according to the present description, said methodcomprising obtaining a transgenic mouse with the entire human TCRαβ geneloci (1.1 and 0.7 Mb), whose T-cells express a diverse human TCRrepertoire that compensates for mouse TCR deficiency, immunizing themouse with peptide of interest, incubating PBMCs obtained from thetransgenic mice with tetramer-phycoerythrin (PE), and isolating the highavidity T-cells by fluorescence activated cell sorting (FACS)-Caliburanalysis.

In one aspect, to obtain T-cells expressing TCRs of the presentdescription, nucleic acids encoding TCR-alpha and/or TCR-beta chains ofthe present description are cloned into expression vectors, such asgamma retrovirus or lentivirus. The recombinant viruses are generatedand then tested for functionality, such as antigen specificity andfunctional avidity. An aliquot of the final product is then used totransduce the target T-cell population (generally purified from patientPBMCs), which is expanded before infusion into the patient. In anotheraspect, to obtain T-cells expressing TCRs of the present description,TCR RNAs are synthesized by techniques known in the art, e.g., in vitrotranscription systems. The in vitro-synthesized TCR RNAs are thenintroduced into primary CD8+ T-cells obtained from healthy donors byelectroporation to re-express tumor specific TCR-alpha and/or TCR-betachains.

To increase the expression, nucleic acids encoding TCRs of the presentdescription may be operably linked to strong promoters, such asretroviral long terminal repeats (LTRs), cytomegalovirus (CMV), murinestem cell virus (MSCV) U3, phosphoglycerate kinase (PGK), β-actin,ubiquitin, and a simian virus 40 (SV40)/CD43 composite promoter,elongation factor (EF)-1a and the spleen focus-forming virus (SFFV)promoter. In a preferred embodiment, the promoter is heterologous to thenucleic acid being expressed.

In addition to strong promoters, TCR expression cassettes of the presentdescription may contain additional elements that can enhance transgeneexpression, including a central polypurine tract (cPPT), which promotesthe nuclear translocation of lentiviral constructs (Follenzi et al.,2000), and the woodchuck hepatitis virus posttranscriptional regulatoryelement (wPRE), which increases the level of transgene expression byincreasing RNA stability (Zufferey et al., 1999).

The alpha and beta chains of a TCR of the present invention may beencoded by nucleic acids located in separate vectors, or may be encodedby polynucleotides located in the same vector.

Achieving high-level TCR surface expression requires that both theTCR-alpha and TCR-beta chains of the introduced TCR be transcribed athigh levels. To do so, the TCR-alpha and TCR-beta chains of the presentdescription may be cloned into bi-cistronic constructs in a singlevector, which has been shown to be capable of overcoming this obstacle.The use of a viral intraribosomal entry site (IRES) between theTCR-alpha and TCR-beta chains results in the coordinated expression ofboth chains, because the TCR-alpha and TCR-beta chains are generatedfrom a single transcript that is broken into two proteins duringtranslation, ensuring that an equal molar ratio of TCR-alpha andTCR-beta chains are produced. (Schmitt et al. 2009).

Nucleic acids encoding TCRs of the present description may be codonoptimized to increase expression from a host cell. Redundancy in thegenetic code allows some amino acids to be encoded by more than onecodon, but certain codons are less “optimal” than others because of therelative availability of matching tRNAs as well as other factors(Gustafsson et al., 2004). Modifying the TCR-alpha and TCR-beta genesequences such that each amino acid is encoded by the optimal codon formammalian gene expression, as well as eliminating mRNA instabilitymotifs or cryptic splice sites, has been shown to significantly enhanceTCR-alpha and TCR-beta gene expression (Scholten et al., 2006).

Furthermore, mispairing between the introduced and endogenous TCR chainsmay result in the acquisition of specificities that pose a significantrisk for autoimmunity. For example, the formation of mixed TCR dimersmay reduce the number of CD3 molecules available to form properly pairedTCR complexes, and therefore can significantly decrease the functionalavidity of the cells expressing the introduced TCR (Kuball et al.,2007).

To reduce mispairing, the C-terminus domain of the introduced TCR chainsof the present description may be modified in order to promoteinterchain affinity, while decreasing the ability of the introducedchains to pair with the endogenous TCR. These strategies may includereplacing the human TCR-alpha and TCR-beta C-terminus domains with theirmurine counterparts (murinized C-terminus domain); generating a secondinterchain disulfide bond in the C-terminus domain by introducing asecond cysteine residue into both the TCR-alpha and TCR-beta chains ofthe introduced TCR (cysteine modification); swapping interactingresidues in the TCR-alpha and TCR-beta chain C-terminus domains(“knob-in-hole”); and fusing the variable domains of the TCR-alpha andTCR-beta chains directly to CD3ζ (CD3ζ fusion). (Schmitt et al. 2009).

In an embodiment, a host cell is engineered to express a TCR of thepresent description. In preferred embodiments, the host cell is a humanT-cell or T-cell progenitor. In some embodiments, the T-cell or T-cellprogenitor is obtained from a cancer patient. In other embodiments, theT-cell or T-cell progenitor is obtained from a healthy donor. Host cellsof the present description can be allogeneic or autologous with respectto a patient to be treated. In one embodiment, the host is a gamma/deltaT-cell transformed to express an alpha/beta TCR.

A “pharmaceutical composition” is a composition suitable foradministration to a human being in a medical setting. Preferably, apharmaceutical composition is sterile and produced according to GMPguidelines.

The pharmaceutical compositions comprise the peptides either in the freeform or in the form of a pharmaceutically acceptable salt (see alsoabove). In an aspect, a pharmaceutically acceptable salt describedherein refers to salts which possess toxicity profiles within a rangethat is acceptable for pharmaceutical applications.

As used herein, “a pharmaceutically acceptable salt” refers to aderivative of the disclosed peptides wherein the peptide is modified bymaking acid or base salts of the agent. For example, acid salts areprepared from the free base (typically wherein the neutral form of thedrug has a neutral —NH2 group) involving reaction with a suitable acid.Suitable acids for preparing acid salts include both organic acids,e.g., acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalicacid, malic acid, malonic acid, succinic acid, maleic acid, fumaricacid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelicacid, methane sulfonic acid, ethane sulfonic acid, p-toluenesulfonicacid, salicylic acid, and the like, as well as inorganic acids, e.g.,hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acidphosphoric acid and the like. Conversely, preparation of basic salts ofacid moieties which may be present on a peptide are prepared using apharmaceutically acceptable base such as sodium hydroxide, potassiumhydroxide, ammonium hydroxide, calcium hydroxide, trimethylamine or thelike. (See, for example, Berge et al. (1977) “Pharmaceutical Salts”, J.Pharm. Sci. 66:1-19, which is herein incorporated by reference in itsentirety).

In an aspect, pharmaceutically acceptable salts may increase thesolubility and/or stability of peptides of described herein. In anotheraspect, pharmaceutical salts described herein may be prepared byconventional means from the corresponding carrier peptide or complex byreacting, for example, the appropriate acid or base with peptides orcomplexes as described herein. In another aspect, the pharmaceuticallyacceptable salts are in crystalline form. In yet another aspect,pharmaceutically acceptable salts may include, for example, thosedescribed in Handbook of Pharmaceutical Salts: Properties, Selection,and Use By P. H. Stahl and C. G. Wermuth (Wiley-VCH 2002) and L. D.Bighley, S. M. Berge, D. C. Monkhouse, in “Encyclopedia ofPharmaceutical Technology”. Eds. J. Swarbrick and J. C. Boylan, Vol. 13,Marcel Dekker, Inc., New York, Basel, Hong Kong 1995, pp. 453-499, eachof these references is herein incorporated by reference in theirentirety.

In an especially preferred embodiment, the pharmaceutical compositionscomprise the peptides as salts of acetic acid (acetates), trifluoroacetates or hydrochloric acid (chlorides).

Preferably, the medicament of the present invention is animmunotherapeutic such as a vaccine. It may be administered directlyinto the patient, into the affected organ or systemically i.d., i.m.,s.c., i.p. and i.v., or applied ex vivo to cells derived from thepatient or a human cell line which are subsequently administered to thepatient, or used in vitro to select a subpopulation of immune cellsderived from the patient, which are then re-administered to the patient.If the nucleic acid is administered to cells in vitro, it may be usefulfor the cells to be transfected so as to co-express immune-stimulatingcytokines, such as interleukin-2. The peptide may be substantially pure,or combined with an immune-stimulating adjuvant (see below) or used incombination with immune-stimulatory cytokines, or be administered with asuitable delivery system, for example liposomes. The peptide may also beconjugated to a suitable carrier such as keyhole limpet haemocyanin(KLH) or mannan (see WO 95/18145 and (Longenecker et al., 1993)). Thepeptide may also be tagged, may be a fusion protein, or may be a hybridmolecule. The peptides whose sequence is given in the present inventionare expected to stimulate CD4 or CD8 T cells. However, stimulation ofCD8 T cells is more efficient in the presence of help provided by CD4T-helper cells. Thus, for MHC Class I epitopes that stimulate CD8 Tcells the fusion partner or sections of a hybrid molecule suitablyprovide epitopes which stimulate CD4-positive T cells. CD4- andCD8-stimulating epitopes are well known in the art and include thoseidentified in the present invention.

In one aspect, the vaccine comprises at least one peptide having theamino acid sequence set forth SEQ ID No. 1 to SEQ ID No. 32, and atleast one additional peptide, preferably two to 50, more preferably twoto 25, even more preferably two to 20 and most preferably two, three,four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen,fourteen, fifteen, sixteen, seventeen or eighteen peptides. Thepeptide(s) may be derived from one or more specific TAAs and may bind toMHC class I molecules.

A further aspect of the invention provides a nucleic acid (for example apolynucleotide) encoding a peptide or peptide variant of the invention.The polynucleotide may be, for example, DNA, cDNA, PNA, RNA orcombinations thereof, either single- and/or double-stranded, or nativeor stabilized forms of polynucleotides, such as, for example,polynucleotides with a phosphorothioate backbone and it may or may notcontain introns so long as it codes for the peptide. Of course, onlypeptides that contain naturally occurring amino acid residues joined bynaturally occurring peptide bonds are encodable by a polynucleotide. Astill further aspect of the invention provides an expression vectorcapable of expressing a polypeptide according to the invention.

A variety of methods have been developed to link polynucleotides,especially DNA, to vectors for example via complementary cohesivetermini. For instance, complementary homopolymer tracts can be added tothe DNA segment to be inserted to the vector DNA.

The vector and DNA segment are then joined by hydrogen bonding betweenthe complementary homopolymeric tails to form recombinant DNA molecules.

Synthetic linkers containing one or more restriction sites provide analternative method of joining the DNA segment to vectors. Syntheticlinkers containing a variety of restriction endonuclease sites arecommercially available from a number of sources including IntemationalBiotechnologies Inc. New Haven, Conn., USA.

A desirable method of modifying the DNA encoding the polypeptide of theinvention employs the polymerase chain reaction as disclosed by Saiki RK, et al. (Saiki et al., 1988).

This method may be used for introducing the DNA into a suitable vector,for example by engineering in suitable restriction sites, or it may beused to modify the DNA in other useful ways as is known in the art. Ifviral vectors are used, pox- or adenovirus vectors are preferred.

The DNA (or in the case of retroviral vectors, RNA) may then beexpressed in a suitable host to produce a polypeptide comprising thepeptide or variant of the invention. Thus, the DNA encoding the peptideor variant of the invention may be used in accordance with knowntechniques, appropriately modified in view of the teachings containedherein, to construct an expression vector, which is then used totransform an appropriate host cell for the expression and production ofthe polypeptide of the invention. Such techniques include thosedisclosed, for example, in U.S. Pat. Nos. 4,440,859, 4,530,901,4,582,800, 4,677,063, 4,678,751, 4,704,362, 4,710,463, 4,757,006,4,766,075, and 4,810,648.

The DNA (or in the case of retroviral vectors, RNA) encoding thepolypeptide constituting the compound of the invention may be joined toa wide variety of other DNA sequences for introduction into anappropriate host. The companion DNA will depend upon the nature of thehost, the manner of the introduction of the DNA into the host, andwhether episomal maintenance or integration is desired.

Generally, the DNA is inserted into an expression vector, such as aplasmid, in proper orientation and correct reading frame for expression.If necessary, the DNA may be linked to the appropriate transcriptionaland translational regulatory control nucleotide sequences recognized bythe desired host, although such controls are generally available in theexpression vector. The vector is then introduced into the host throughstandard techniques. Generally, not all of the hosts will be transformedby the vector. Therefore, it will be necessary to select for transformedhost cells. One selection technique involves incorporating into theexpression vector a DNA sequence, with any necessary control elements,that codes for a selectable trait in the transformed cell, such asantibiotic resistance.

Alternatively, the gene for such selectable trait can be on anothervector, which is used to co-transform the desired host cell.

Host cells that have been transformed by the recombinant DNA of theinvention are then cultured for a sufficient time and under appropriateconditions known to those skilled in the art in view of the teachingsdisclosed herein to permit the expression of the polypeptide, which canthen be recovered.

Many expression systems are known, including bacteria (for example E.coli and Bacillus subtilis), yeasts (for example Saccharomycescerevisiae), filamentous fungi (for example Aspergillus spec.), plantcells, animal cells and insect cells. Preferably, the system can bemammalian cells such as CHO cells available from the ATCC Cell BiologyCollection.

A typical mammalian cell vector plasmid for constitutive expressioncomprises the CMV or SV40 promoter with a suitable poly A tail and aresistance marker, such as neomycin. One example is pSVL available fromPharmacia, Piscataway, N.J., USA. An example of an inducible mammalianexpression vector is pMSG, also available from Pharmacia. Useful yeastplasmid vectors are pRS403-406 and pRS413-416 and are generallyavailable from Stratagene Cloning Systems, La Jolla, Calif. 92037, USA.Plasmids pRS403, pRS404, pRS405 and pRS406 are Yeast Integratingplasmids (YIps) and incorporate the yeast selectable markers HIS3, TRP1,LEU2 and URA3. Plasmids pRS413-416 are Yeast Centromere plasmids (Ycps).CMV promoter-based vectors (for example from Sigma-Aldrich) providetransient or stable expression, cytoplasmic expression or secretion, andN-terminal or C-terminal tagging in various combinations of FLAG,3xFLAG, c-myc or MAT. These fusion proteins allow for detection,purification and analysis of recombinant protein. Dual-tagged fusionsprovide flexibility in detection.

The strong human cytomegalovirus (CMV) promoter regulatory region drivesconstitutive protein expression levels as high as 1 mg/L in COS cells.For less potent cell lines, protein levels are typically −0.1 mg/L. Thepresence of the SV40 replication origin will result in high levels ofDNA replication in SV40 replication permissive COS cells. CMV vectors,for example, can contain the pMB1 (derivative of pBR322) origin forreplication in bacterial cells, the b-lactamase gene for ampicillinresistance selection in bacteria, hGH polyA, and the fl origin. Vectorscontaining the pre-pro-trypsin leader (PPT) sequence can direct thesecretion of FLAG fusion proteins into the culture medium forpurification using ANTI-FLAG antibodies, resins, and plates. Othervectors and expression systems are well known in the art for use with avariety of host cells.

In another embodiment two or more peptides or peptide variants of theinvention are encoded and thus expressed in a successive order (similarto “beads on a string” constructs). In doing so, the peptides or peptidevariants may be linked or fused together by stretches of linker aminoacids, such as for example LLLLLL, or may be linked without anyadditional peptide(s) between them. These constructs can also be usedfor cancer therapy, and may induce immune responses both involving MHC Iand MHC II.

The present invention also relates to a host cell transformed with apolynucleotide vector construct of the present invention. The host cellcan be either prokaryotic or eukaryotic.

Bacterial cells may be preferred prokaryotic host cells in somecircumstances and typically are a strain of E. coli such as, forexample, the E. coli strains DH5 available from Bethesda ResearchLaboratories Inc., Bethesda, Md., USA, and RR1 available from theAmerican Type Culture Collection (ATCC) of Rockville, Md., USA (No ATCC31343).

Preferred eukaryotic host cells include yeast, insect and mammaliancells, preferably vertebrate cells such as those from a mouse, rat,monkey or human fibroblastic and colon cell lines. Yeast host cellsinclude YPH499, YPH500 and YPH501, which are generally available fromStratagene Cloning Systems, La Jolla, Calif. 92037, USA. Preferredmammalian host cells include Chinese hamster ovary (CHO) cells availablefrom the ATCC as CCL61, NIH Swiss mouse embryo cells NIH/3T3 availablefrom the ATCC as CRL 1658, monkey kidney-derived COS-1 cells availablefrom the ATCC as CRL 1650 and 293 cells which are human embryonic kidneycells. Preferred insect cells are Sf9 cells which can be transfectedwith baculovirus expression vectors. An overview regarding the choice ofsuitable host cells for expression can be found in, for example, thetextbook of Paulina Balbas and Argelia Lorence “Methods in MolecularBiology Recombinant Gene Expression, Reviews and Protocols,” Part One,Second Edition, ISBN 978-1-58829-262-9, and other literature known tothe person of skill.

Transformation of appropriate cell hosts with a DNA construct of thepresent invention is accomplished by well-known methods that typicallydepend on the type of vector used.

With regard to transformation of prokaryotic host cells, see, forexample, Cohen et al. (Cohen et al., 1972) and (Green and Sambrook,2012). Transformation of yeast cells is described in Sherman et al.(Sherman et al., 1986). The method of Beggs (Beggs, 1978) is alsouseful. With regard to vertebrate cells, reagents useful in transfectingsuch cells, for example calcium phosphate and DEAE-dextran or liposomeformulations, are available from Stratagene Cloning Systems, or LifeTechnologies Inc., Gaithersburg, Md. 20877, USA. Electroporation is alsouseful for transforming and/or transfecting cells and is well known inthe art for transforming yeast cell, bacterial cells, insect cells andvertebrate cells.

Successfully transformed cells, i.e. cells that contain a DNA constructof the present invention, can be identified by well-known techniquessuch as PCR. Alternatively, the presence of the protein in thesupernatant can be detected using antibodies.

It will be appreciated that certain host cells of the invention areuseful in the preparation of the peptides of the invention, for examplebacterial, yeast and insect cells. However, other host cells may beuseful in certain therapeutic methods. For example, antigen-presentingcells, such as dendritic cells, may usefully be used to express thepeptides of the invention such that they may be loaded into appropriateMHC molecules. Thus, the current invention provides a host cellcomprising a nucleic acid or an expression vector according to theinvention.

In a preferred embodiment, the host cell is an antigen presenting cell,in particular a dendritic cell or antigen presenting cell. APCs loadedwith a recombinant fusion protein containing prostatic acid phosphatase(PAP) were approved by the U.S. Food and Drug Administration (FDA) onApr. 29, 2010, to treat asymptomatic or minimally symptomatic metastaticHRPC (Sipuleucel-T) (Rini et al., 2006; Small et al., 2006).

A further aspect of the invention provides a method of producing apeptide or its variant, the method comprising culturing a host cell andisolating the peptide from the host cell or its culture medium.

In another embodiment the peptide, the nucleic acid or the expressionvector of the invention are used in medicine. For example, the peptideor its variant may be prepared for intravenous (i.v.) injection,sub-cutaneous (s.c.) injection, intradermal (i.d.) injection,intraperitoneal (i.p.) injection, intramuscular (i.m.) injection.Preferred methods of peptide injection include s.c., i.d., i.p., i.m.,and i.v. Preferred methods of DNA injection include i.d., i.m., s.c.,i.p. and i.v. Doses of e.g. between 50 μg and 1.5 mg, preferably 125 μgto 500 μg, of peptide or DNA may be given and will depend on therespective peptide or DNA. Dosages of this range were successfully usedin previous trials (Walter et al., 2012).

The polynucleotide used for active vaccination may be substantiallypure, or contained in a suitable vector or delivery system. The nucleicacid may be DNA, cDNA, PNA, RNA or a combination thereof. Methods fordesigning and introducing such a nucleic acid are well known in the art.An overview is provided by e.g. Teufel et al. (Teufel et al., 2005).Polynucleotide vaccines are easy to prepare, but the mode of action ofthese vectors in inducing an immune response is not fully understood.Suitable vectors and delivery systems include viral DNA and/or RNA, suchas systems based on adenovirus, vaccinia virus, retroviruses, herpesvirus, adeno-associated virus or hybrids containing elements of morethan one virus. Non-viral delivery systems include cationic lipids andcationic polymers and are well known in the art of DNA delivery.Physical delivery, such as via a “gene-gun” may also be used. Thepeptide or peptides encoded by the nucleic acid may be a fusion protein,for example with an epitope that stimulates T cells for the respectiveopposite CDR as noted above.

The medicament of the invention may also include one or more adjuvants.Adjuvants are substances that non-specifically enhance or potentiate theimmune response (e.g., immune responses mediated by CD8-positive T cellsand helper-T (TH) cells to an antigen, and would thus be considereduseful in the medicament of the present invention. Suitable adjuvantsinclude, but are not limited to, 1018 ISS, aluminum salts, AMPLIVAX®,AS15, BCG, CP-870,893, CpG7909, CyaA, dSLIM, flagellin or TLR5 ligandsderived from flagellin, FLT3 ligand, GM-CSF, IC30, IC31, Imiquimod(ALDARA®), resiquimod, ImuFact IMP321, Interleukins as IL-2, IL-13,IL-21, Interferon-alpha or -beta, or pegylated derivatives thereof, ISPatch, ISS, ISCOMATRIX, ISCOMs, Juvlmmune®, LipoVac, MALP2, MF59,monophosphoryl lipid A, Montanide IMS 1312, Montanide ISA 206, MontanideISA 50V, Montanide ISA-51, water-in-oil and oil-in-water emulsions,OK-432, OM-174, OM-197-MP-EC, ONTAK, OspA, PepTel® vector system,poly(lactid co-glycolid) [PLG]-based and dextran microparticles,talactoferrin SRL172, Virosomes and other Virus-like particles, YF-17D,VEGF trap, R848, beta-glucan, Pam3Cys, Aquila's QS21 stimulon, which isderived from saponin, mycobacterial extracts and synthetic bacterialcell wall mimics, and other proprietary adjuvants such as Ribi's Detox,Quil, or Superfos. Adjuvants such as Freund's or GM-CSF are preferred.Several immunological adjuvants (e.g., MF59) specific for dendriticcells and their preparation have been described previously (Allison andKrummel, 1995). Also, cytokines may be used. Several cytokines have beendirectly linked to influencing dendritic cell migration to lymphoidtissues (e.g., TNF−), accelerating the maturation of dendritic cellsinto efficient antigen-presenting cells for T-lymphocytes (e.g., GM-CSF,IL-1 and IL-4) (U.S. Pat. No. 5,849,589, specifically incorporatedherein by reference in its entirety) and acting as immunoadjuvants(e.g., IL-12, IL-15, IL-23, IL-7, IFN-alpha. IFN-beta) (Gabrilovich etal., 1996).

CpG immunostimulatory oligonucleotides have also been reported toenhance the effects of adjuvants in a vaccine setting. Without beingbound by theory, CpG oligonucleotides act by activating the innate(non-adaptive) immune system via Toll-like receptors (TLR), mainly TLR9.CpG triggered TLR9 activation enhances antigen-specific humoral andcellular responses to a wide variety of antigens, including peptide orprotein antigens, live or killed viruses, dendritic cell vaccines,autologous cellular vaccines and polysaccharide conjugates in bothprophylactic and therapeutic vaccines. More importantly it enhancesdendritic cell maturation and differentiation, resulting in enhancedactivation of TH1 cells and strong cytotoxic T-lymphocyte (CTL)generation, even in the absence of CD4 T cell help. The TH1 bias inducedby TLR9 stimulation is maintained even in the presence of vaccineadjuvants such as alum or incomplete Freund's adjuvant (IFA) thatnormally promote a TH2 bias. CpG oligonucleotides show even greateradjuvant activity when formulated or co-administered with otheradjuvants or in formulations such as microparticles, nanoparticles,lipid emulsions or similar formulations, which are especially necessaryfor inducing a strong response when the antigen is relatively weak. Theyalso accelerate the immune response and enable the antigen doses to bereduced by approximately two orders of magnitude, with comparableantibody responses to the full-dose vaccine without CpG in someexperiments (Krieg, 2006). U.S. Pat. No. 6,406,705 B1 describes thecombined use of CpG oligonucleotides, non-nucleic acid adjuvants and anantigen to induce an antigen-specific immune response. A CpG TLR9antagonist is dSLIM (double Stem Loop Immunomodulator) by Mologen(Berlin, Germany) which is a preferred component of the pharmaceuticalcomposition of the present invention. Other TLR binding molecules suchas RNA binding TLR 7, TLR 8 and/or TLR 9 may also be used.

Other examples for useful adjuvants include, but are not limited tochemically modified CpGs (e.g. CpR, Idera), dsRNA analogues such asPoly(I:C) and derivates thereof (e.g. AmpliGen®, Hiltonol®, poly-(ICLC),poly(IC-R), poly(I:C12U), non-CpG bacterial DNA or RNA as well asimmunoactive small molecules and antibodies such as cyclophosphamide,Sunitinib, Bevacizumab®, Celebrex, NCX-4016, sildenafil, tadalafil,vardenafil, Sorafenib, temozolomide, temsirolimus, XL-999, CP-547632,pazopanib, VEGF Trap, ZD2171, AZD2171, anti-CTLA4, other antibodiestargeting key structures of the immune system (e.g. anti-CD40,anti-TGFbeta, anti-TNFalpha receptor) and SC58175, which may acttherapeutically and/or as an adjuvant. The amounts and concentrations ofadjuvants and additives useful in the context of the present inventioncan readily be determined by the skilled artisan without undueexperimentation.

Preferred adjuvants are anti-CD40, imiquimod, resiquimod, GM-CSF,cyclophosphamide, sunitinib, bevacizumab, interferon-alpha, CpGoligonucleotides and derivates, poly-(I:C) and derivates, RNA,sildenafil, and particulate formulations with PLG or virosomes.

In a preferred embodiment, the pharmaceutical composition according tothe invention the adjuvant is selected from the group consisting ofcolony-stimulating factors, such as Granulocyte Macrophage ColonyStimulating Factor (GM-CSF, sargramostim), cyclophosphamide, imiquimod,resiquimod, and interferon-alpha.

In a preferred embodiment, the pharmaceutical composition according tothe invention the adjuvant is selected from the group consisting ofcolony-stimulating factors, such as Granulocyte Macrophage ColonyStimulating Factor (GM-CSF, sargramostim), cyclophosphamide, imiquimodand resiquimod. In a preferred embodiment of the pharmaceuticalcomposition according to the invention, the adjuvant iscyclophosphamide, imiquimod or resiquimod. Even more preferred adjuvantsare Montanide IMS 1312, Montanide ISA 206, Montanide ISA 50V, MontanideISA-51, poly-ICLC (Hiltonoli) and anti-CD40 mAB, or combinationsthereof.

This composition is used for parenteral administration, such assubcutaneous, intradermal, intramuscular or oral administration. Forthis, the peptides and optionally other molecules are dissolved orsuspended in a pharmaceutically acceptable, preferably aqueous carrier.In an aspect, peptides or other molecules described herein may becombined with an aquous carrier. In an aspect, the aquous carrier isselected from ion exchangers, alumina, aluminum stearate, magnesiumstearate, lecithin, serum proteins, such as human serum albumin, buffersubstances such as phosphates, glycine, sorbic acid, potassium sorbate,partial glyceride mixtures of saturated vegetable fatty acids, salts orelectrolytes, such as protamine sulfate, disodium hydrogen phosphate,dicalcium phosphate, potassium hydrogen phosphate, sodium chloride, zincsalts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone,polyvinylpyrrolidone-vinyl acetate, cellulose-based substances (e.g.,microcrystalline cellulose, hydroxypropyl methylcellulose, hydroxypropylmethylcellulose acetate succinate, hydroxypropyl methylcellulosePhthalate), starch, lactose monohydrate, mannitol, trehalose sodiumlauryl sulfate, and crosscarmellose sodium, polyethylene glycol, sodiumcarboxymethylcellulose, polyacrylates, polymethacrylate, waxes,polyethylene-polyoxypropylene-block polymers, polyethylene glycol andwool fat.

In an aspect, the aquous carrier contains multiple components, such aswater together with a non-water carrier component, such as thosecomponents described herein. In another aspect, the aquous carrier iscapable of imparting improved properties when combined with a peptide orother molecule described herein, for example, improved solubility,efficiacy, and/or improved immunotherapy. In addition, the compositioncan contain excipients, such as buffers, binding agents, blastingagents, diluents, flavors, lubricants, etc. A “pharmaceuticallyacceptable diluent,” for example, may include solvents, bulking agents,stabilizing agents, dispersion media, coatings, antibacterial andantifungal agents, isotonic and absorption delaying agents, and the likewhich are physiologically compatible. Examples of pharmaceuticallyacceptable diluents include one or more of saline, phosphate bufferedsaline, dextrose, glycerol, ethanol, and the like as well ascombinations thereof. In many cases it will be preferable to include oneor more isotonic agents, for example, sugars such as trehalose andsucrose, polyalcohols such as mannitol, sorbitol, or sodium chloride inthe composition. Pharmaceutically acceptable substances such as wettingor minor amounts of auxiliary substances such as wetting or emulsifyingagents, preservatives or buffers, are also within the scope of thepresent invention.In addition, the composition can contain excipients,such as buffers, binding agents, blasting agents, diluents, flavors,lubricants, etc. The peptides can also be administered together withimmune stimulating substances, such as cytokines. An extensive listingof excipients that can be used in such a composition, can be, forexample, taken from A. Kibbe, Handbook of Pharmaceutical Excipients(Kibbe, 2000).

The composition can be used for a prevention, prophylaxis and/or therapyof adenomatous or cancerous diseases. Exemplary formulations can befound in, for example, EP2112253.

It is important to realize that the immune response triggered by thevaccine according to the invention attacks the cancer in differentcell-stages and different stages of development. Furthermore, differentcancer associated signaling pathways are attacked.

This is an advantage over vaccines that address only one or few targets,which may cause the tumor to easily adapt to the attack (tumor escape).Furthermore, not all individual tumors express the same pattern ofantigens. Therefore, a combination of several tumor-associated peptidesensures that every single tumor bears at least some of the targets.

The composition is designed in such a way that each tumor is expected toexpress several of the antigens and cover several independent pathwaysnecessary for tumor growth and maintenance. Thus, the vaccine can easilybe used “off-the-shelf” for a larger patient population. This means thata pre-selection of patients to be treated with the vaccine can berestricted to HLA typing, does not require any additional biomarkerassessments for antigen expression, but it is still ensured that severaltargets are simultaneously attacked by the induced immune response,which is important for efficacy (Banchereau et al., 2001; Walter et al.,2012).

As used herein, the term “scaffold” refers to a molecule thatspecifically binds to an (e.g. antigenic) determinant. In oneembodiment, a scaffold is able to direct the entity to which it isattached (e.g. a (second) antigen binding moiety) to a target site, forexample to a specific type of tumor cell or tumor stroma bearing theantigenic determinant (e.g. the complex of a peptide with MHC, accordingto the application at hand). In another embodiment, a scaffold is ableto activate signaling through its target antigen, for example a T cellreceptor complex antigen. Scaffolds include but are not limited toantibodies and fragments thereof, antigen binding domains of anantibody, comprising an antibody heavy chain variable region and anantibody light chain variable region, binding proteins comprising atleast one ankyrin repeat motif and single domain antigen binding (SDAB)molecules, aptamers, (soluble) TCRs and (modified) cells such asallogenic or autologous T cells. To assess whether a molecule is ascaffold binding to a target, binding assays can be performed.

“Specific” binding means that the scaffold binds the peptide-MHC-complexof interest better than other naturally occurring peptide-MHC-complexes,to an extent that a scaffold armed with an active molecule that is ableto kill a cell bearing the specific target is not able to kill anothercell without the specific target but presenting another peptide-MHCcomplex(es). Binding to other peptide-MHC complexes is irrelevant if thepeptide of the cross-reactive peptide-MHC is not naturally occurring,i.e. not derived from the human HLA-peptidome. Tests to assess targetcell killing are well known in the art. They should be performed usingtarget cells (primary cells or cell lines) with unaltered peptide-MHCpresentation, or cells loaded with peptides such that naturallyoccurring peptide-MHC levels are reached.

Each scaffold can comprise a labelling which provides that the boundscaffold can be detected by determining the presence or absence of asignal provided by the label. For example, the scaffold can be labelledwith a fluorescent dye or any other applicable cellular marker molecule.Such marker molecules are well known in the art. For example, afluorescence-labelling, for example provided by a fluorescence dye, canprovide a visualization of the bound aptamer by fluorescence or laserscanning microscopy or flow cytometry.

Each scaffold can be conjugated with a second active molecule such asfor example IL-21, anti-CD3, and anti-CD28.

For further information on polypeptide scaffolds see for example thebackground section of WO 2014/071978A1 and the references cited therein.

The present invention further relates to aptamers. Aptamers (see forexample WO 2014/191359 and the literature as cited therein) are shortsingle-stranded nucleic acid molecules, which can fold into definedthree-dimensional structures and recognize specific target structures.They have appeared to be suitable altematives for developing targetedtherapies. Aptamers have been shown to selectively bind to a variety ofcomplex targets with high affinity and specificity.

Aptamers recognizing cell surface located molecules have been identifiedwithin the past decade and provide means for developing diagnostic andtherapeutic approaches. Since aptamers have been shown to possess almostno toxicity and immunogenicity they are promising candidates forbiomedical applications. Indeed aptamers, for example prostate-specificmembrane-antigen recognizing aptamers, have been successfully employedfor targeted therapies and shown to be functional in xenograft in vivomodels. Furthermore, aptamers recognizing specific tumor cell lines havebeen identified.

DNA aptamers can be selected to reveal broad-spectrum recognitionproperties for various cancer cells, and particularly those derived fromsolid tumors, while non-tumorigenic and primary healthy cells are notrecognized. If the identified aptamers recognize not only a specifictumor sub-type but rather interact with a series of tumors, this rendersthe aptamers applicable as so-called broad-spectrum diagnostics andtherapeutics.

Further, investigation of cell-binding behavior with flow cytometryshowed that the aptamers revealed very good apparent affinities that arewithin the nanomolar range.

Aptamers are useful for diagnostic and therapeutic purposes. Further, itcould be shown that some of the aptamers are taken up by tumor cells andthus can function as molecular vehicles for the targeted delivery ofanti-cancer agents such as siRNA into tumor cells.

Aptamers can be selected against complex targets such as cells andtissues and complexes of the peptides comprising, preferably consistingof, a sequence according to any of SEQ ID NO 1 to SEQ ID NO 32,according to the invention at hand with the MHC molecule, using thecell-SELEX (Systematic Evolution of Ligands by Exponential enrichment)technique.

The peptides of the present invention can be used to generate anddevelop specific antibodies against MHC/peptide complexes. These can beused for therapy, targeting toxins or radioactive substances to thediseased tissue. Another use of these antibodies can be targetingradionuclides to the diseased tissue for imaging purposes such as PET.

This use can help to detect small metastases or to determine the sizeand precise localization of diseased tissues.

Therefore, it is a further aspect of the invention to provide a methodfor producing a recombinant antibody specifically binding to a humanmajor histocompatibility complex (MHC) class I or II being complexedwith a HLA-restricted antigen, the method comprising: immunizing agenetically engineered non-human mammal comprising cells expressing saidhuman major histocompatibility complex (MHC) class I or II with asoluble form of a MHC class I or II molecule being complexed with saidHLA-restricted antigen; isolating mRNA molecules from antibody producingcells of said non-human mammal; producing a phage display librarydisplaying protein molecules encoded by said mRNA molecules; andisolating at least one phage from said phage display library, said atleast one phage displaying said antibody specifically binding to saidhuman major histocompatibility complex (MHC) class I or II beingcomplexed with said HLA-restricted antigen.

It is a further aspect of the invention to provide an antibody thatspecifically binds to a human major histocompatibility complex (MHC)class I or II being complexed with a HLA-restricted antigen, wherein theantibody preferably is a polyclonal antibody, monoclonal antibody,bi-specific antibody and/or a chimeric antibody.

Respective methods for producing such antibodies and single chain classI major histocompatibility complexes, as well as other tools for theproduction of these antibodies are disclosed in WO 03/068201, WO2004/084798, WO 01/72768, WO 03/070752, and in publications (Cohen etal., 2003a; Cohen et al., 2003b; Denkberg et al., 2003), which for thepurposes of the present invention are all explicitly incorporated byreference in their entireties.

Preferably, the antibody is binding with a binding affinity of below 20nanomolar, preferably of below 10 nanomolar, to the complex, which isalso regarded as “specific” in the context of the present invention.

The present invention relates to a peptide comprising a sequence that isselected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 32, ora variant thereof which is at least 88% homologous (preferablyidentical) to SEQ ID NO: 1 to SEQ ID NO: 32 or a variant thereof thatinduces T cells cross-reacting with said peptide, wherein said peptideis not the underlying full-length polypeptide.

The present invention further relates to a peptide comprising a sequencethat is selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO:32 or a variant thereof which is at least 88% homologous (preferablyidentical) to SEQ ID NO: 1 to SEQ ID NO: 32, wherein said peptide orvariant has an overall length of between 8 and 100, preferably between 8and 30, and most preferred between 8 and 14 amino acids.

The present invention further relates to the peptides according to theinvention that have the ability to bind to a molecule of the human majorhistocompatibility complex (MHC) class-I or -II.

The present invention further relates to the peptides according to theinvention wherein the peptide consists or consists essentially of anamino acid sequence according to SEQ ID NO: 1 to SEQ ID NO: 32.

The present invention further relates to the peptides according to theinvention, wherein the peptide is (chemically) modified and/or includesnon-peptide bonds.

The present invention further relates to the peptides according to theinvention, wherein the peptide is part of a fusion protein, inparticular comprising N-terminal amino acids of the HLA-DRantigen-associated invariant chain (li), or wherein the peptide is fusedto (or into) an antibody, such as, for example, an antibody that isspecific for dendritic cells.

The present invention further relates to a nucleic acid, encoding thepeptides according to the invention, provided that the peptide is notthe complete (full) human protein.

The present invention further relates to the nucleic acid according tothe invention that is DNA, cDNA, PNA, RNA or combinations thereof.

The present invention further relates to an expression vector capable ofexpressing a nucleic acid according to the present invention.

The present invention further relates to a peptide according to thepresent invention, a nucleic acid according to the present invention oran expression vector according to the present invention for use inmedicine, in particular in the treatment of gallbladder cancer andcholangiocarcinoma.

The present invention further relates to a host cell comprising anucleic acid according to the invention or an expression vectoraccording to the invention.

The present invention further relates to the host cell according to thepresent invention that is an antigen presenting cell, and preferably adendritic cell.

The present invention further relates to a method of producing a peptideaccording to the present invention, said method comprising culturing thehost cell according to the present invention, and isolating the peptidefrom said host cell or its culture medium.

The present invention further relates to the method according to thepresent invention, wherein the antigen is loaded onto class I or II MHCmolecules expressed on the surface of a suitable antigen-presenting cellby contacting a sufficient amount of the antigen with anantigen-presenting cell.

The present invention further relates to the method according to theinvention, wherein the antigen-presenting cell comprises an expressionvector capable of expressing said peptide containing SEQ ID NO: 1 to SEQID NO: 32 or said variant amino acid sequence.

The present invention further relates to activated T cells, produced bythe method according to the present invention, wherein said T cellsselectively recognizes a cell which aberrantly expresses a polypeptidecomprising an amino acid sequence according to the present invention.

The present invention further relates to a method of killing targetcells in a patient which target cells aberrantly express a polypeptidecomprising any amino acid sequence according to the present invention,the method comprising administering to the patient an effective numberof T cells as according to the present invention.

The present invention further relates to the use of any peptidedescribed, a nucleic acid according to the present invention, anexpression vector according to the present invention, a cell accordingto the present invention, or an activated cytotoxic T lymphocyteaccording to the present invention as a medicament or in the manufactureof a medicament. The present invention further relates to a useaccording to the present invention, wherein the medicament is activeagainst cancer.

The present invention further relates to a use according to theinvention, wherein the medicament is a vaccine. The present inventionfurther relates to a use according to the invention, wherein themedicament is active against cancer.

The present invention further relates to a use according to theinvention, wherein said cancer cells are gallbladder cancer andcholangiocarcinoma cells or other solid or hematological tumor cellssuch as acute myeloid leukemia, melanoma, small cell lung cancer,non-small cell lung cancer, non-Hodgkin lymphoma, chronic lymphocyticleukemia, pancreatic cancer, liver cancer, ovarian cancer, head and neckcancer, urinary bladder cancer, breast cancer, and kidney cancer.

The present invention further relates to particular marker proteins andbiomarkers based on the peptides according to the present invention,herein called “targets” that can be used in the diagnosis and/orprognosis of gallbladder cancer and cholangiocarcinoma. The presentinvention also relates to the use of these novel targets for cancertreatment.

The term “antibody” or “antibodies” is used herein in a broad sense andincludes both polyclonal and monoclonal antibodies. In addition tointact or “full” immunoglobulin molecules, also included in the term“antibodies” are fragments (e.g. CDRs, Fv, Fab and Fc fragments) orpolymers of those immunoglobulin molecules and humanized versions ofimmunoglobulin molecules, as long as they exhibit any of the desiredproperties (e.g., specific binding of a gallbladder cancer andcholangiocarcinoma marker (poly)peptide, delivery of a toxin to agallbladder cancer and cholangiocarcinoma cell expressing a cancermarker gene at an increased level, and/or inhibiting the activity of agallbladder cancer and cholangiocarcinoma marker polypeptide) accordingto the invention.

Whenever possible, the antibodies of the invention may be purchased fromcommercial sources. The antibodies of the invention may also begenerated using well-known methods. The skilled artisan will understandthat either full length gallbladder cancer and cholangiocarcinoma markerpolypeptides or fragments thereof may be used to generate the antibodiesof the invention. A polypeptide to be used for generating an antibody ofthe invention may be partially or fully purified from a natural source,or may be produced using recombinant DNA techniques.

For example, a cDNA encoding a peptide according to the presentinvention, such as a peptide according to SEQ ID NO: 1 to SEQ ID NO: 32polypeptide, or a variant or fragment thereof, can be expressed inprokaryotic cells (e.g., bacteria) or eukaryotic cells (e.g., yeast,insect, or mammalian cells), after which the recombinant protein can bepurified and used to generate a monoclonal or polyclonal antibodypreparation that specifically bind the gallbladder cancer andcholangiocarcinoma marker polypeptide used to generate the antibodyaccording to the invention.

One of skill in the art will realize that the generation of two or moredifferent sets of monoclonal or polyclonal antibodies maximizes thelikelihood of obtaining an antibody with the specificity and affinityrequired for its intended use (e.g., ELISA, immunohistochemistry, invivo imaging, immunotoxin therapy). The antibodies are tested for theirdesired activity by known methods, in accordance with the purpose forwhich the antibodies are to be used (e.g., ELISA, immunohistochemistry,immunotherapy, etc.; for further guidance on the generation and testingof antibodies, see, e.g., Greenfield, 2014 (Greenfield, 2014)). Forexample, the antibodies may be tested in ELISA assays or, Western blots,immunohistochemical staining of formalin-fixed cancers or frozen tissuesections. After their initial in vitro characterization, antibodiesintended for therapeutic or in vivo diagnostic use are tested accordingto known clinical testing methods.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a substantially homogeneous population of antibodies,i.e.; the individual antibodies comprising the population are identicalexcept for possible naturally occurring mutations that may be present inminor amounts. The monoclonal antibodies herein specifically include“chimeric” antibodies in which a portion of the heavy and/or light chainis identical with or homologous to corresponding sequences in antibodiesderived from a particular species or belonging to a particular antibodyclass or subclass, while the remainder of the chain(s) is identical withor homologous to corresponding sequences in antibodies derived fromanother species or belonging to another antibody class or subclass, aswell as fragments of such antibodies, so long as they exhibit thedesired antagonistic activity (U.S. Pat. No. 4,816,567, which is herebyincorporated in its entirety).

Monoclonal antibodies of the invention may be prepared using hybridomamethods. In a hybridoma method, a mouse or other appropriate host animalis typically immunized with an immunizing agent to elicit lymphocytesthat produce or are capable of producing antibodies that willspecifically bind to the immunizing agent. Alternatively, thelymphocytes may be immunized in vitro.

The monoclonal antibodies may also be made by recombinant DNA methods,such as those described in U.S. Pat. No. 4,816,567. DNA encoding themonoclonal antibodies of the invention can be readily isolated andsequenced using conventional procedures (e.g., by using oligonucleotideprobes that are capable of binding specifically to genes encoding theheavy and light chains of murine antibodies).

In vitro methods are also suitable for preparing monovalent antibodies.Digestion of antibodies to produce fragments thereof, particularly Fabfragments, can be accomplished using routine techniques known in theart. For instance, digestion can be performed using papain. Examples ofpapain digestion are described in WO 94/29348 and U.S. Pat. No.4,342,566. Papain digestion of antibodies typically produces twoidentical antigen binding fragments, called Fab fragments, each with asingle antigen binding site, and a residual Fc fragment. Pepsintreatment yields a F(ab′)2 fragment and a pFc′ fragment.

The antibody fragments, whether attached to other sequences or not, canalso include insertions, deletions, substitutions, or other selectedmodifications of particular regions or specific amino acids residues,provided the activity of the fragment is not significantly altered orimpaired compared to the non-modified antibody or antibody fragment.These modifications can provide for some additional property, such as toremove/add amino acids capable of disulfide bonding, to increase itsbio-longevity, to alter its secretory characteristics, etc. In any case,the antibody fragment must possess a bioactive property, such as bindingactivity, regulation of binding at the binding domain, etc.

Functional or active regions of the antibody may be identified bymutagenesis of a specific region of the protein, followed by expressionand testing of the expressed polypeptide.

Such methods are readily apparent to a skilled practitioner in the artand can include site-specific mutagenesis of the nucleic acid encodingthe antibody fragment.

The antibodies of the invention may further comprise humanizedantibodies or human antibodies. Humanized forms of non-human (e.g.,murine) antibodies are chimeric immunoglobulins, immunoglobulin chainsor fragments thereof (such as Fv, Fab, Fab′ or other antigen-bindingsubsequences of antibodies) which contain minimal sequence derived fromnon-human immunoglobulin. Humanized antibodies include humanimmunoglobulins (recipient antibody) in which residues from acomplementary determining region (CDR) of the recipient are replaced byresidues from a CDR of a non-human species (donor antibody) such asmouse, rat or rabbit having the desired specificity, affinity andcapacity. In some instances, Fv framework (FR) residues of the humanimmunoglobulin are replaced by corresponding non-human residues.Humanized antibodies may also comprise residues which are found neitherin the recipient antibody nor in the imported CDR or frameworksequences. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the CDR regions correspond to thoseof a non-human immunoglobulin and all or substantially all of the FRregions are those of a human immunoglobulin consensus sequence. Thehumanized antibody optimally also will comprise at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin.

Methods for humanizing non-human antibodies are well known in the art.Generally, a humanized antibody has one or more amino acid residuesintroduced into it from a source which is non-human. These non-humanamino acid residues are often referred to as “import” residues, whichare typically taken from an “import” variable domain.

Humanization can be essentially performed by substituting rodent CDRs orCDR sequences for the corresponding sequences of a human antibody.Accordingly, such “humanized” antibodies are chimeric antibodies (U.S.Pat. No. 4,816,567), wherein substantially less than an intact humanvariable domain has been substituted by the corresponding sequence froma non-human species. In practice, humanized antibodies are typicallyhuman antibodies in which some CDR residues and possibly some FRresidues are substituted by residues from analogous sites in rodentantibodies.

Transgenic animals (e.g., mice) that are capable, upon immunization, ofproducing a full repertoire of human antibodies in the absence ofendogenous immunoglobulin production can be employed. For example, ithas been described that the homozygous deletion of the antibody heavychain joining region gene in chimeric and germ-line mutant mice resultsin complete inhibition of endogenous antibody production. Transfer ofthe human germ-line immunoglobulin gene array in such germ-line mutantmice will result in the production of human antibodies upon antigenchallenge. Human antibodies can also be produced in phage displaylibraries.

Antibodies of the invention are preferably administered to a subject ina pharmaceutically acceptable carrier. Typically, an appropriate amountof a pharmaceutically-acceptable salt is used in the formulation torender the formulation isotonic. Examples of thepharmaceutically-acceptable carrier include saline, Ringer's solutionand dextrose solution. The pH of the solution is preferably from about 5to about 8, and more preferably from about 7 to about 7.5. Furthercarriers include sustained release preparations such as semipermeablematrices of solid hydrophobic polymers containing the antibody, whichmatrices are in the form of shaped articles, e.g., films, liposomes ormicroparticles. It will be apparent to those persons skilled in the artthat certain carriers may be more preferable depending upon, forinstance, the route of administration and concentration of antibodybeing administered.

The antibodies can be administered to the subject, patient, or cell byinjection (e.g., intravenous, intraperitoneal, subcutaneous,intramuscular), or by other methods such as infusion that ensure itsdelivery to the bloodstream in an effective form. The antibodies mayalso be administered by intratumoral or peritumoral routes, to exertlocal as well as systemic therapeutic effects. Local or intravenousinjection is preferred.

Effective dosages and schedules for administering the antibodies may bedetermined empirically, and making such determinations is within theskill in the art. Those skilled in the art will understand that thedosage of antibodies that must be administered will vary depending on,for example, the subject that will receive the antibody, the route ofadministration, the particular type of antibody used and other drugsbeing administered.

A typical daily dosage of the antibody used alone might range from about1 (μg/kg to up to 100 mg/kg of body weight or more per day, depending onthe factors mentioned above.

Following administration of an antibody, preferably for treatinggallbladder cancer and cholangiocarcinoma, the efficacy of thetherapeutic antibody can be assessed in various ways well known to theskilled practitioner. For instance, the size, number, and/ordistribution of cancer in a subject receiving treatment may be monitoredusing standard tumor imaging techniques. A therapeutically-administeredantibody that arrests tumor growth, results in tumor shrinkage, and/orprevents the development of new tumors, compared to the disease coursethat would occurs in the absence of antibody administration, is anefficacious antibody for treatment of cancer.

It is a further aspect of the invention to provide a method forproducing a soluble T-cell receptor (sTCR) recognizing a specificpeptide-MHC complex. Such soluble T-cell receptors can be generated fromspecific T-cell clones, and their affinity can be increased bymutagenesis targeting the complementarity-determining regions. For thepurpose of T-cell receptor selection, phage display can be used (US2010/0113300, (Liddy et al., 2012)). For the purpose of stabilization ofT-cell receptors during phage display and in case of practical use asdrug, alpha and beta chain can be linked e.g. by non-native disulfidebonds, other covalent bonds (single-chain T-cell receptor), or bydimerization domains (Boulter et al., 2003; Card et al., 2004; Willcoxet al., 1999). The T-cell receptor can be linked to toxins, drugs,cytokines (see, for example, US 2013/0115191), and domains recruitingeffector cells such as an anti-CD3 domain, etc., in order to executeparticular functions on target cells. Moreover, it could be expressed inT cells used for adoptive transfer. Further information can be found inWO 2004/033685A1 and WO 2004/074322A1. A combination of sTCRs isdescribed in WO 2012/056407A1. Further methods for the production aredisclosed in WO 2013/057586A1.

In addition, the peptides and/or the TCRs or antibodies or other bindingmolecules of the present invention can be used to verify a pathologist'sdiagnosis of a cancer based on a biopsied sample.

The antibodies or TCRs may also be used for in vivo diagnostic assays.Generally, the antibody is labeled with a radionucleotide (such as¹¹¹In, ⁹⁹Tc, ¹⁴C, ¹³¹I, ³H, ³²P or ³⁵S) so that the tumor can belocalized using immunoscintiography. In one embodiment, antibodies orfragments thereof bind to the extracellular domains of two or moretargets of a protein selected from the group consisting of theabove-mentioned proteins, and the affinity value (Kd) is less than 1×10μM.

Antibodies for diagnostic use may be labeled with probes suitable fordetection by various imaging methods. Methods for detection of probesinclude, but are not limited to, fluorescence, light, confocal andelectron microscopy; magnetic resonance imaging and spectroscopy;fluoroscopy, computed tomography and positron emission tomography.

Suitable probes include, but are not limited to, fluorescein, rhodamine,eosin and other fluorophores, radioisotopes, gold, gadolinium and otherlanthanides, paramagnetic iron, fluorine-18 and other positron-emittingradionuclides. Additionally, probes may be bi- or multi-functional andbe detectable by more than one of the methods listed. These antibodiesmay be directly or indirectly labeled with said probes. Attachment ofprobes to the antibodies includes covalent attachment of the probe,incorporation of the probe into the antibody, and the covalentattachment of a chelating compound for binding of probe, amongst otherswell recognized in the art. For immunohistochemistry, the disease tissuesample may be fresh or frozen or may be embedded in paraffin and fixedwith a preservative such as formalin. The fixed or embedded sectioncontains the sample are contacted with a labeled primary antibody andsecondary antibody, wherein the antibody is used to detect theexpression of the proteins in situ.

Another aspect of the present invention includes an in vitro method forproducing activated T cells, the method comprising contacting in vitro Tcells with antigen loaded human MHC molecules expressed on the surfaceof a suitable antigen-presenting cell for a period of time sufficient toactivate the T cell in an antigen specific manner, wherein the antigenis a peptide according to the invention. Preferably, a sufficient amountof the antigen is used with an antigen-presenting cell.

Preferably the mammalian cell lacks or has a reduced level or functionof the TAP peptide transporter. Suitable cells that lack the TAP peptidetransporter include T2, RMA-S and Drosophila cells. TAP is thetransporter associated with antigen processing.

The human peptide loading deficient cell line T2 is available from theAmerican Type Culture Collection, 12301 Parklawn Drive, Rockville, Md.20852, USA under Catalogue No CRL 1992; the Drosophila cell lineSchneider line 2 is available from the ATCC under Catalogue No CRL19863; the mouse RMA-S cell line is described in Ljunggren et al.(Ljunggren and Karre, 1985).

Preferably, before transfection the host cell expresses substantially noMHC class I molecules. It is also preferred that the stimulator cellexpresses a molecule important for providing a co-stimulatory signal forT-cells such as any of B7.1, B7.2, ICAM-1 and LFA 3. The nucleic acidsequences of numerous MHC class I molecules and of the co-stimulatormolecules are publicly available from the GenBank and EMBL databases.

In case of a MHC class I epitope being used as an antigen, the T cellsare CD8-positive T cells.

If an antigen-presenting cell is transfected to express such an epitope,preferably the cell comprises an expression vector capable of expressinga peptide containing SEQ ID NO: 1 to SEQ ID NO: 32, or a variant aminoacid sequence thereof.

A number of other methods may be used for generating T cells in vitro.For example, autologous tumor-infiltrating lymphocytes can be used inthe generation of CTL.

Plebanski et al. (Plebanski et al., 1995) made use of autologousperipheral blood lymphocytes (PLBs) in the preparation of T cells.Furthermore, the production of autologous T cells by pulsing dendriticcells with peptide or polypeptide, or via infection with recombinantvirus is possible. Also, B cells can be used in the production ofautologous T cells. In addition, macrophages pulsed with peptide orpolypeptide, or infected with recombinant virus, may be used in thepreparation of autologous T cells. S.

Walter et al. (Walter et al., 2003) describe the in vitro priming of Tcells by using artificial antigen presenting cells (aAPCs), which isalso a suitable way for generating T cells against the peptide ofchoice. In the present invention, aAPCs were generated by the couplingof preformed MHC: peptide complexes to the surface of polystyreneparticles (microbeads) by biotin: streptavidin biochemistry. This systempermits the exact control of the MHC density on aAPCs, which allows toselectively eliciting high- or low-avidity antigen-specific T cellresponses with high efficiency from blood samples. Apart from MHC:peptide complexes, aAPCs should carry other proteins with co-stimulatoryactivity like anti-CD28 antibodies coupled to their surface.Furthermore, such aAPC-based systems often require the addition ofappropriate soluble factors, e. g. cytokines, like interleukin-12.

Allogeneic cells may also be used in the preparation of T cells and amethod is described in detail in WO 97/26328, incorporated herein byreference. For example, in addition to Drosophila cells and T2 cells,other cells may be used to present antigens such as CHO cells,baculovirus-infected insect cells, bacteria, yeast, andvaccinia-infected target cells. In addition, plant viruses may be used(see, for example, Porta et al. (Porta et al., 1994) which describes thedevelopment of cowpea mosaic virus as a high-yielding system for thepresentation of foreign peptides.

The activated T cells that are directed against the peptides of theinvention are useful in therapy. Thus, a further aspect of the inventionprovides activated T cells obtainable by the foregoing methods of theinvention.

Activated T cells, which are produced by the above method, willselectively recognize a cell that aberrantly expresses a polypeptidethat comprises an amino acid sequence of SEQ ID NO: 1 to SEQ ID NO 32.

Preferably, the T cell recognizes the cell by interacting through itsTCR with the HLA/peptide-complex (for example, binding). The T cells areuseful in a method of killing target cells in a patient whose targetcells aberrantly express a polypeptide comprising an amino acid sequenceof the invention wherein the patient is administered an effective numberof the activated T cells. The T cells that are administered to thepatient may be derived from the patient and activated as described above(i.e. they are autologous T cells). Alternatively, the T cells are notfrom the patient but are from another individual. Of course, it ispreferred if the individual is a healthy individual. By “healthyindividual” the inventors mean that the individual is generally in goodhealth, preferably has a competent immune system and, more preferably,is not suffering from any disease that can be readily tested for, anddetected.

In vivo, the target cells for the CD8-positive T cells according to thepresent invention can be cells of the tumor (which sometimes express MHCclass II) and/or stromal cells surrounding the tumor (tumor cells)(which sometimes also express MHC class II; (Dengjel et al., 2006)).

The T cells of the present invention may be used as active ingredientsof a therapeutic composition. Thus, the invention also provides a methodof killing target cells in a patient whose target cells aberrantlyexpress a polypeptide comprising an amino acid sequence of theinvention, the method comprising administering to the patient aneffective number of T cells as defined above.

By “aberrantly expressed” the inventors also mean that the polypeptideis over-expressed compared to levels of expression in normal tissues orthat the gene is silent in the tissue from which the tumor is derivedbut in the tumor, it is expressed. By “over-expressed” the inventorsmean that the polypeptide is present at a level at least 1.2-fold ofthat present in normal tissue; preferably at least 2-fold, and morepreferably at least 5-fold or 10-fold the level present in normaltissue.

T cells may be obtained by methods known in the art, e.g. thosedescribed above.

Protocols for this so-called adoptive transfer of T cells are well knownin the art. Reviews can be found in: Gattioni et al. and Morgan et al.(Gattinoni et al., 2006; Morgan et al., 2006).

Another aspect of the present invention includes the use of the peptidescomplexed with MHC to generate a T-cell receptor whose nucleic acid iscloned and is introduced into a host cell, preferably a T cell. Thisengineered T cell can then be transferred to a patient for therapy ofcancer.

Any molecule of the invention, i.e. the peptide, nucleic acid, antibody,expression vector, cell, activated T cell, T-cell receptor or thenucleic acid encoding it, is useful for the treatment of disorders,characterized by cells escaping an immune response. Therefore, anymolecule of the present invention may be used as medicament or in themanufacture of a medicament. The molecule may be used by itself orcombined with other molecule(s) of the invention or (a) knownmolecule(s).

The present invention further provides a medicament that is useful intreating cancer, in particular gallbladder cancer and cholangiocarcinomaand other malignancies.

The present invention is further directed at a kit comprising:

(a) a container containing a pharmaceutical composition as describedabove, in solution or in lyophilized form;(b) optionally a second container containing a diluent or reconstitutingsolution for the lyophilized formulation; and(c) optionally, instructions for (i) use of the solution or (ii)reconstitution and/or use of the lyophilized formulation.

The kit may further comprise one or more of (iii) a buffer, (iv) adiluent, (v) a filter, (vi) a needle, or (v) a syringe. The container ispreferably a bottle, a vial, a syringe or test tube; and it may be amulti-use container. The pharmaceutical composition is preferablylyophilized.

Kits of the present invention preferably comprise a lyophilizedformulation of the present invention in a suitable container andinstructions for its reconstitution and/or use. Suitable containersinclude, for example, bottles, vials (e.g. dual chamber vials), syringes(such as dual chamber syringes) and test tubes. The container may beformed from a variety of materials such as glass or plastic. Preferablythe kit and/or container contain/s instructions on or associated withthe container that indicates directions for reconstitution and/or use.For example, the label may indicate that the lyophilized formulation isto be reconstituted to peptide concentrations as described above. Thelabel may further indicate that the formulation is useful or intendedfor subcutaneous administration.

The container holding the formulation may be a multi-use vial, whichallows for repeat administrations (e.g., from 2-6 administrations) ofthe reconstituted formulation. The kit may further comprise a secondcontainer comprising a suitable diluent (e.g., sodium bicarbonatesolution).

Upon mixing of the diluent and the lyophilized formulation, the finalpeptide concentration in the reconstituted formulation is preferably atleast 0.15 mg/mL/peptide (=75 μg) and preferably not more than 3mg/mL/peptide (=1500 μg). The kit may further include other materialsdesirable from a commercial and user standpoint, including otherbuffers, diluents, filters, needles, syringes, and package inserts withinstructions for use.

Kits of the present invention may have a single container that containsthe formulation of the pharmaceutical compositions according to thepresent invention with or without other components (e.g., othercompounds or pharmaceutical compositions of these other compounds) ormay have distinct container for each component.

Preferably, kits of the invention include a formulation of the inventionpackaged for use in combination with the co-administration of a secondcompound (such as adjuvants (e.g. GM-CSF), a chemotherapeutic agent, anatural product, a hormone or antagonist, an anti-angiogenesis agent orinhibitor, an apoptosis-inducing agent or a chelator) or apharmaceutical composition thereof. The components of the kit may bepre-complexed or each component may be in a separate distinct containerprior to administration to a patient. The components of the kit may beprovided in one or more liquid solutions, preferably, an aqueoussolution, more preferably, a sterile aqueous solution. The components ofthe kit may also be provided as solids, which may be converted intoliquids by addition of suitable solvents, which are preferably providedin another distinct container.

The container of a therapeutic kit may be a vial, test tube, flask,bottle, syringe, or any other means of enclosing a solid or liquid.Usually, when there is more than one component, the kit will contain asecond vial or other container, which allows for separate dosing. Thekit may also contain another container for a pharmaceutically acceptableliquid. Preferably, a therapeutic kit will contain an apparatus (e.g.,one or more needles, syringes, eye droppers, pipette, etc.), whichenables administration of the agents of the invention that arecomponents of the present kit.

The present formulation is one that is suitable for administration ofthe peptides by any acceptable route such as oral (enteral), nasal,ophthal, subcutaneous, intradermal, intramuscular, intravenous ortransdermal. Preferably, the administration is s.c., and most preferablyi.d. administration may be by infusion pump.

Since the peptides of the invention were isolated from gallbladdercancer and cholangiocarcinoma, the medicament of the invention ispreferably used to treat gallbladder cancer and cholangiocarcinoma.

The present invention further relates to a method for producing apersonalized pharmaceutical for an individual patient comprisingmanufacturing a pharmaceutical composition comprising at least onepeptide selected from a warehouse of pre-screened TUMAPs, wherein the atleast one peptide used in the pharmaceutical composition is selected forsuitability in the individual patient. In one embodiment, thepharmaceutical composition is a vaccine. The method could also beadapted to produce T cell clones for down-stream applications, such asTCR isolations, or soluble antibodies, and other treatment options.

A “personalized pharmaceutical” shall mean specifically tailoredtherapies for one individual patient that will only be used for therapyin such individual patient, including actively personalized cancervaccines and adoptive cellular therapies using autologous patienttissue.

As used herein, the term “warehouse” shall refer to a group or set ofpeptides that have been pre-screened for immunogenicity and/orover-presentation in a particular tumor type. The term “warehouse” isnot intended to imply that the particular peptides included in thevaccine have been pre-manufactured and stored in a physical facility,although that possibility is contemplated. It is expressly contemplatedthat the peptides may be manufactured de novo for each individualizedvaccine produced, or may be pre-manufactured and stored. The warehouse(e.g. in the form of a database) is composed of tumor-associatedpeptides which were highly overexpressed in the tumor tissue ofgallbladder cancer and cholangiocarcinoma patients with various HLA-AHLA-B and HLA-C alleles. It may contain MHC class I and MHC class IIpeptides or elongated MHC class I peptides. In addition to the tumorassociated peptides collected from several gallbladder cancer andcholangiocarcinoma tissues, the warehouse may contain HLA-A*02 andHLA-A*24 marker peptides. These peptides allow comparison of themagnitude of T-cell immunity induced by TUMAPS in a quantitative mannerand hence allow important conclusion to be drawn on the capacity of thevaccine to elicit anti-tumor responses.

Secondly, they function as important positive control peptides derivedfrom a “non-self” antigen in the case that any vaccine-induced T-cellresponses to TUMAPs derived from “self” antigens in a patient are notobserved. And thirdly, it may allow conclusions to be drawn, regardingthe status of immunocompetence of the patient.

TUMAPs for the warehouse are identified by using an integratedfunctional genomics approach combining gene expression analysis, massspectrometry, and T-cell immunology (XPresident 6). The approach assuresthat only TUMAPs truly present on a high percentage of tumors but not oronly minimally expressed on normal tissue, are chosen for furtheranalysis. For initial peptide selection, gallbladder cancer andcholangiocarcinoma samples from patients and blood from healthy donorswere analyzed in a stepwise approach:

1. HLA ligands from the malignant material were identified by massspectrometry2. Genome-wide messenger ribonucleic acid (mRNA) expression analysis wasused to identify genes over-expressed in the malignant tissue(gallbladder cancer and cholangiocarcinoma) compared with a range ofnormal organs and tissues3. Identified HLA ligands were compared to gene expression data.Peptides over-presented or selectively presented on tumor tissue,preferably encoded by selectively expressed or over-expressed genes asdetected in step 2 were considered suitable TUMAP candidates for amulti-peptide vaccine.4. Literature research was performed in order to identify additionalevidence supporting the relevance of the identified peptides as TUMAPs5. The relevance of over-expression at the mRNA level was confirmed byredetection of selected TUMAPs from step 3 on tumor tissue and lack of(or infrequent) detection on healthy tissues.6. In order to assess, whether an induction of in vivo T-cell responsesby the selected peptides may be feasible, in vitro immunogenicity assayswere performed using human T cells from healthy donors as well as fromgallbladder cancer and cholangiocarcinoma patients.

In an aspect, the peptides are pre-screened for immunogenicity beforebeing included in the warehouse. By way of example, and not limitation,the immunogenicity of the peptides included in the warehouse isdetermined by a method comprising in vitro T-cell priming throughrepeated stimulations of CD8+ T cells from healthy donors withartificial antigen presenting cells loaded with peptide/MHC complexesand anti-CD28 antibody.

This method is preferred for rare cancers and patients with a rareexpression profile. In contrast to multi-peptide cocktails with a fixedcomposition as currently developed, the warehouse allows a significantlyhigher matching of the actual expression of antigens in the tumor withthe vaccine. Selected single or combinations of several “off-the-shelf”peptides will be used for each patient in a multitarget approach. Intheory, an approach based on selection of e.g. 5 different antigenicpeptides from a library of 50 would already lead to approximately 17million possible drug product (DP) compositions.

In an aspect, the peptides are selected for inclusion in the vaccinebased on their suitability for the individual patient based on themethod according to the present invention as described herein, or asbelow.

The HLA phenotype, transcriptomic and peptidomic data is gathered fromthe patient's tumor material, and blood samples to identify the mostsuitable peptides for each patient containing “warehouse” andpatient-unique (i.e. mutated) TUMAPs. Those peptides will be chosen,which are selectively or over-expressed in the patients' tumor and,where possible, show strong in vitro immunogenicity if tested with thepatients' individual PBMCs.

Preferably, the peptides included in the vaccine are identified by amethod comprising: (a) identifying tumor-associated peptides (TUMAPs)presented by a tumor sample from the individual patient; (b) comparingthe peptides identified in (a) with a warehouse (database) of peptidesas described above; and (c) selecting at least one peptide from thewarehouse (database) that correlates with a tumor-associated peptideidentified in the patient. For example, the TUMAPs presented by thetumor sample are identified by: (a1) comparing expression data from thetumor sample to expression data from a sample of normal tissuecorresponding to the tissue type of the tumor sample to identifyproteins that are over-expressed or aberrantly expressed in the tumorsample; and (a2) correlating the expression data with sequences of MHCligands bound to MHC class I and/or class II molecules in the tumorsample to identify MHC ligands derived from proteins over-expressed oraberrantly expressed by the tumor. Preferably, the sequences of MHCligands are identified by eluting bound peptides from MHC moleculesisolated from the tumor sample, and sequencing the eluted ligands.Preferably, the tumor sample and the normal tissue are obtained from thesame patient.

In addition to, or as an alternative to, selecting peptides using awarehousing (database) model, TUMAPs may be identified in the patient denovo, and then included in the vaccine. As one example, candidate TUMAPsmay be identified in the patient by (a1) comparing expression data fromthe tumor sample to expression data from a sample of normal tissuecorresponding to the tissue type of the tumor sample to identifyproteins that are over-expressed or aberrantly expressed in the tumorsample; and (a2) correlating the expression data with sequences of MHCligands bound to MHC class I and/or class II molecules in the tumorsample to identify MHC ligands derived from proteins over-expressed oraberrantly expressed by the tumor. As another example, proteins may beidentified containing mutations that are unique to the tumor samplerelative to normal corresponding tissue from the individual patient, andTUMAPs can be identified that specifically target the mutation. Forexample, the genome of the tumor and of corresponding normal tissue canbe sequenced by whole genome sequencing: For discovery of non-synonymousmutations in the protein-coding regions of genes, genomic DNA and RNAare extracted from tumor tissues and normal non-mutated genomic germlineDNA is extracted from peripheral blood mononuclear cells (PBMCs). Theapplied NGS approach is confined to the re-sequencing of protein codingregions (exome re-sequencing). For this purpose, exonic DNA from humansamples is captured using vendor-supplied target enrichment kits,followed by sequencing with e.g. a HiSeq2000 (Illumina). Additionally,tumor mRNA is sequenced for direct quantification of gene expression andvalidation that mutated genes are expressed in the patients' tumors. Theresultant millions of sequence reads are processed through softwarealgorithms. The output list contains mutations and gene expression.Tumor-specific somatic mutations are determined by comparison with thePBMC-derived germline variations and prioritized.

The de novo identified peptides can then be tested for immunogenicity asdescribed above for the warehouse, and candidate TUMAPs possessingsuitable immunogenicity are selected for inclusion in the vaccine.

In one exemplary embodiment, the peptides included in the vaccine areidentified by: (a) identifying tumor-associated peptides (TUMAPs)presented by a tumor sample from the individual patient by the method asdescribed above; (b) comparing the peptides identified in a) with awarehouse of peptides that have been prescreened for immunogenicity andoverpresentation in tumors as compared to corresponding normal tissue;(c) selecting at least one peptide from the warehouse that correlateswith a tumor-associated peptide identified in the patient; and (d)optionally, selecting at least one peptide identified de novo in (a)confirming its immunogenicity.

In one exemplary embodiment, the peptides included in the vaccine areidentified by: (a) identifying tumor-associated peptides (TUMAPs)presented by a tumor sample from the individual patient; and (b)selecting at least one peptide identified de novo in (a) and confirmingits immunogenicity.

Once the peptides for a personalized peptide based vaccine are selected,the vaccine is produced. The vaccine preferably is a liquid formulationconsisting of the individual peptides dissolved in between 20-40% DMSO,preferably about 30-35% DMSO, such as about 33% DMSO.

Each peptide to be included into a product is dissolved in DMSO. Theconcentration of the single peptide solutions has to be chosen dependingon the number of peptides to be included into the product. The singlepeptide-DMSO solutions are mixed in equal parts to achieve a solutioncontaining all peptides to be included in the product with aconcentration of −2.5 mg/ml per peptide. The mixed solution is thendiluted 1:3 with water for injection to achieve a concentration of 0.826mg/ml per peptide in 33% DMSO. The diluted solution is filtered througha 0.22 μm sterile filter. The final bulk solution is obtained.

Final bulk solution is filled into vials and stored at −20° C. untiluse. One vial contains 700 μL solution, containing 0.578 mg of eachpeptide. Of this, 500 μL (approx. 400 μg per peptide) will be appliedfor intradermal injection.

In addition to being useful for treating cancer, the peptides of thepresent invention are also useful as diagnostics. Since the peptideswere generated from gallbladder cancer and cholangiocarcinoma cells andsince it was determined that these peptides are not or at lower levelspresent in normal tissues, these peptides can be used to diagnose thepresence of a cancer.

The presence of claimed peptides on tissue biopsies in blood samples canassist a pathologist in diagnosis of cancer. Detection of certainpeptides by means of antibodies, mass spectrometry or other methodsknown in the art can tell the pathologist that the tissue sample ismalignant or inflamed or generally diseased, or can be used as abiomarker for gallbladder cancer and cholangiocarcinoma. Presence ofgroups of peptides can enable classification or sub-classification ofdiseased tissues.

The detection of peptides on diseased tissue specimen can enable thedecision about the benefit of therapies involving the immune system,especially if T-lymphocytes are known or expected to be involved in themechanism of action. Loss of MHC expression is a well describedmechanism by which infected of malignant cells escapeimmuno-surveillance.

Thus, presence of peptides shows that this mechanism is not exploited bythe analyzed cells.

The peptides of the present invention might be used to analyzelymphocyte responses against those peptides such as T cell responses orantibody responses against the peptide or the peptide complexed to MHCmolecules. These lymphocyte responses can be used as prognostic markersfor decision on further therapy steps. These responses can also be usedas surrogate response markers in immunotherapy approaches aiming toinduce lymphocyte responses by different means, e.g. vaccination ofprotein, nucleic acids, autologous materials, adoptive transfer oflymphocytes. In gene therapy settings, lymphocyte responses againstpeptides can be considered in the assessment of side effects. Monitoringof lymphocyte responses might also be a valuable tool for follow-upexaminations of transplantation therapies, e.g. for the detection ofgraft versus host and host versus graft diseases.

The present invention will now be described in the following exampleswhich describe preferred embodiments thereof, and with reference to theaccompanying figures, nevertheless, without being limited thereto. Forthe purposes of the present invention, all references as cited hereinare incorporated by reference in their entireties.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A to 1G show the over-presentation of various peptides in normaltissues (white bars) and gallbladder cancer and cholangiocarcinoma(black bars). FIG. 1A: Gene symbol: MON2, Peptide: AVMTDLPVI, (SEQ IDNO.: 16), Tissues from left to right: 6 adipose tissues, 8 adrenalglands, 24 blood cells, 17 blood vessels, 10 bone marrows, 15 brains, 8breasts, 4 cartilages, 7 esophagi, 2 eyes, 16 hearts, 19 kidneys, 21large intestines, 25 livers, 49 lungs, 8 lymph nodes, 13 nerves, 3ovaries, 11 pancreases, 6 parathyroid glands, 1 peritoneum, 6 pituitaryglands, 7 placentas, 1 pleura, 3 prostates, 7 salivary glands, 10skeletal muscles, 12 skins, 6 small intestines, 12 spleens, 5 stomachs,7 testes, 2 thymi, 2 thyroid glands, 14 tracheas, 7 ureters, 8 urinarybladders, 6 uteri, 3 gallbladders, 17 gallbladder cancer andcholangiocarcinoma samples. The peptide has additionally been detectedon 5/18 acute myeloid leukemias, 7/48 benign prostatic hyperplasias,8/18 breast cancers, 4/17 chronic lymphocytic leukemias, 1/29 colorectalcancers, 3/34 brain cancers, 4/21 liver cancers, 4/10 head and neckcancers, 4/18 melanomas, 12/20 non-Hodgkin lymphomas, 10/90 non-smallcell lung cancers, 6/20 ovarian cancers, 2/18 esophageal cancers, 3/19pancreatic cancers, 3/23 kidney cancers, 6/17 small cell lung cancers,3/15 urinary bladder cancers, and 6/16 uterus cancers.

FIG. 1B: Gene symbol: CDC25B, Peptide: ISAPLVKTL (SEQ ID NO.: 24),Tissues from left to right: 6 adipose tissues, 8 adrenal glands, 24blood cells, 17 blood vessels, 10 bone marrows, 15 brains, 8 breasts, 4cartilages, 7 esophagi, 2 eyes, 16 hearts, 19 kidneys, 21 largeintestines, 25 livers, 49 lungs, 8 lymph nodes, 13 nerves, 3 ovaries, 11pancreases, 6 parathyroid glands, 1 peritoneum, 6 pituitary glands, 7placentas, 1 pleura, 3 prostates, 7 salivary glands, 10 skeletalmuscles, 12 skins, 6 small intestines, 12 spleens, 5 stomachs, 7 testes,2 thymi, 2 thyroid glands, 14 tracheas, 7 ureters, 8 urinary bladders, 6uteri, 3 gallbladders, 17 gallbladder cancer and cholangiocarcinomasamples. The peptide has additionally been detected on 1/20 ovariancancers. FIG. 1C: Gene symbol: RNF19A, Peptide: NLSETASTMAL (SEQ ID NO.:25), Tissues from left to right: 6 adipose tissues, 8 adrenal glands, 24blood cells, 17 blood vessels, 10 bone marrows, 15 brains, 8 breasts, 4cartilages, 7 esophagi, 2 eyes, 16 hearts, 19 kidneys, 21 largeintestines, 25 livers, 49 lungs, 8 lymph nodes, 13 nerves, 3 ovaries, 11pancreases, 6 parathyroid glands, 1 peritoneum, 6 pituitary glands, 7placentas, 1 pleura, 3 prostates, 7 salivary glands, 10 skeletalmuscles, 12 skins, 6 small intestines, 12 spleens, 5 stomachs, 7 testes,2 thymi, 2 thyroid glands, 14 tracheas, 7 ureters, 8 urinary bladders, 6uteri, 3 gallbladders, 17 gallbladder cancer and cholangiocarcinomasamples. The peptide has additionally been detected on 1/21 livercancers, 4/90 non-small cell lung cancers, 1/17 small cell lung cancers,1/20 non-Hodgkin lymphomas, 1/20 ovarian cancers, and 1/15 urinarybladder cancers. FIG. 1D: Gene symbol: MEGF6, Peptide: VLQDELPQL (SEQ IDNO.: 20), Samples from left to right: 12 normal tissues (1 esophagus, 2livers, 3 lungs, 1 rectum, 1 skin, 1 small intestine, 1 spleen, 2tracheas), 45 cancer tissues (3 bile duct cancers, 1 breast cancer, 1esophageal cancer, 4 gallbladder cancers, 5 head and neck cancers, 3kidney cancers, 4 leukocytic leukemia cancers, 2 liver cancers, 9 lungcancers, 2 lymph node cancers, 1 myeloid cell cancer, 2 ovarian cancers,3 pancreas cancers, 4 prostate cancers, 1 urinary bladder cancer). FIGS.1E through 1G show the over-presentation of various peptides indifferent cancer tissues (black dots). Upper part: Median MS signalintensities from technical replicate measurements are plotted as dotsfor single HLA-A*02 positive normal (grey dots) and tumor samples (blackdots) on which the peptide was detected. Tumor and normal samples aregrouped according to organ of origin, and box-and-whisker plotsrepresent median, 25th and 75th percentile (box), and minimum andmaximum (whiskers) of normalized signal intensities over multiplesamples. Normal organs are ordered according to risk categories (bloodcells, blood vessels, brain, liver, lung: high risk, grey dots;reproductive organs, breast, prostate: low risk, grey dots; all otherorgans: medium risk; grey dots). Lower part: The relative peptidedetection frequency in every organ is shown as spine plot. Numbers belowthe panel indicate number of samples on which the peptide was detectedout of the total number of samples analyzed for each organ (N=526 fornormal samples, N=562 for tumor samples). If the peptide has beendetected on a sample but could not be quantified for technical reasons,the sample is included in this representation of detection frequency,but no dot is shown in the upper part of the figure. Tissues (from leftto right): Normal samples: blood cells; bloodvess (blood vessels);brain; heart; liver; lung; adipose (adipose tissue); adren.gl. (adrenalgland); bile duct; bladder; BM (bone marrow); cartilage; esoph(esophagus); eye; gallb (gallbladder); head&neck; kidney; large_int(large intestine); LN (lymph node); nerve; pancreas; parathyr(parathyroid gland); perit (peritoneum); pituit (pituitary); pleura;skel.mus (skeletal muscle); skin; small_int (small intestine); spleen;stomach; thyroid; trachea; ureter; breast; ovary; placenta; prostate;testis; thymus; uterus. Tumor samples: AML: acute myeloid leukemia;BRCA: breast cancer; CCC: cholangiocellular carcinoma; CLL: chroniclymphocytic leukemia; CRC: colorectal cancer; GBC: gallbladder cancer;GBM: glioblastoma; GC: gastric cancer; GEJC: stomach cardia esophagus,cancer; HCC: hepatocellular carcinoma; HNSCC: head-and-neck cancer; MEL:melanoma; NHL: non-hodgkin lymphoma; NSCLC: non-small cell lung cancer;OC: ovarian cancer; OSCAR: esophageal cancer; PACA: pancreatic cancer;PRCA: prostate cancer; RCC: renal cell carcinoma; SCLC: small cell lungcancer; UBC: urinary bladder carcinoma; UEC: uterine and endometrialcancer. FIG. 1E) Gene symbol: PLEKHM3, Peptide: VLYDNTQLQL (SEQ ID NO.:17), FIG. 1F) Gene symbol: ILF2, Peptide: TAQTLVRIL (SEQ ID NO.: 26),FIG. 1G) Gene symbol: STAM, Peptide: FASEVSNVL (SEQ ID NO.: 29).

FIGS. 2A to 2C show exemplary expression profiles of source genes of thepresent invention that are highly over-expressed or exclusivelyexpressed in gallbladder cancer and cholangiocarcinoma in a panel ofnormal tissues (white bars) and 10 gallbladder cancer andcholangiocarcinoma samples (black bars). Tissues from left to right: 6arteries, 2 blood cells, 5 brains, 3 hearts, 3 livers, 3 lungs, 2 veins,1 adipose tissue, 1 adrenal gland, 1 bile duct, 5 bone marrows, 1cartilage, 1 colon, 1 esophagus, 2 eyes, 2 gallbladders, 2 salivaryglands, 1 kidney, 6 lymph nodes, 4 pancreases, 1 parathyroid gland, 2peripheral nerves, 2 peritoneums, 2 pituitary glands, 2 pleuras, 1rectum, 2 skeletal muscles, 1 skin, 1 small intestine, 1 spleen, 1stomach, 1 thyroid gland, 7 tracheas, 2 ureters, 1 urinary bladder, 1breast, 5 ovaries, 5 placentas, 1 prostate, 1 testis, 1 thymus, 1uterus, 11 gallbladder cancer and cholangiocarcinoma samples. FIG. 2A)Gene symbol: CYP2W1, FIG. 2B) Gene symbol: PKHD1, FIG. 2C) Gene symbol:SUCNR1.

FIG. 3 shows exemplary immunogenicity data: flow cytometry results afterpeptide-specific multimer staining.

FIGS. 4A-4C show exemplary results of peptide-specific in vitro CD8+ Tcell responses of a healthy HLA-A*02+ donor. CD8+ T cells were primedusing artificial APCs coated with anti-CD28 mAb and HLA-A*02 in complexwith SeqID No 13 peptide (FIG. 4A, left panel), SeqID No 16 peptide(FIG. 4B, left panel) and SeqID No 25 peptide (FIG. 4C, left panel),respectively. After three cycles of stimulation, the detection ofpeptide-reactive cells was performed by 2D multimer staining withA*02/SeqID No 13 (FIG. 4A), A*02/SeqID No 16 (FIG. 4B) or A*02/SeqID No25 (FIG. 4C). Right panels (FIGS. 4A, 4B and 4C) show control stainingof cells stimulated with irrelevant A*02/peptide complexes. Viablesinglet cells were gated for CD8+ lymphocytes. Boolean gates helpedexcluding false-positive events detected with multimers specific fordifferent peptides. Frequencies of specific multimer+ cells among CD8+lymphocytes are indicated.

EXAMPLES Example 1

Identification and Quantitation of Tumor Associated Peptides Presentedon the Cell Surface

Tissue Samples

Patients' tumor tissues were obtained from: Conversant HealthcareSystems Inc., Huntsville, Ala., USA), ProteoGenex Inc. (Culver City,Calif., USA), Tissue Solutions Ltd (Glasgow, UK), University HospitalTübingen (Tübingen, Germany). Normal tissues were obtained from Asterand(Detroit, Mich., USA & Royston, Herts, UK), Bio-Options Inc. (Brea,Calif., USA), BioServe (Beltsville, Md., USA), Capital BioScience Inc.(Rockville, Md., USA), Geneticist Inc. (Glendale, Calif., USA), KyotoPrefectural University of Medicine (KPUM) (Kyoto, Japan), ProteoGenexInc. (Culver City, Calif., USA), Tissue Solutions Ltd (Glasgow, UK),University Hospital Geneva (Geneva, Switzerland), University HospitalHeidelberg (Heidelberg, Germany), University Hospital Munich (Munich,Germany), University Hospital Tübingen (Tübingen, Germany).

Written informed consents of all patients had been given before surgeryor autopsy. Tissues were shock-frozen immediately after excision andstored until isolation of TUMAPs at −70° C. or below.

Isolation of HLA Peptides from Tissue Samples

HLA peptide pools from shock-frozen tissue samples were obtained byimmune precipitation from solid tissues according to a slightly modifiedprotocol (Falk et al., 1991; Seeger et al., 1999) using theHLA-A*02-specific antibody BB7.2, the HLA-A, —B, C-specific antibodyW6/32, CNBr-activated sepharose, acid treatment, and ultrafiltration.

Mass Spectrometry Analyses

The HLA peptide pools as obtained were separated according to theirhydrophobicity by reversed-phase chromatography (nanoAcquity UPLCsystem, Waters) and the eluting peptides were analyzed in LTQ-velos andfusion hybrid mass spectrometers (ThermoElectron) equipped with an ESIsource. Peptide pools were loaded directly onto the analyticalfused-silica micro-capillary column (75 μm i.d.×250 mm) packed with 1.7μm C18 reversed-phase material (Waters) applying a flow rate of 400 nLper minute. Subsequently, the peptides were separated using a two-step180 minute-binary gradient from 10% to 33% B at a flow rate of 300 nLper minute. The gradient was composed of Solvent A (0.1% formic acid inwater) and solvent B (0.1% formic acid in acetonitrile). A gold coatedglass capillary (PicoTip, New Objective) was used for introduction intothe nanoESI source. The LTQ-Orbitrap mass spectrometers were operated inthe data-dependent mode using a TOP5 strategy. In brief, a scan cyclewas initiated with a full scan of high mass accuracy in the orbitrap(R=30 000), which was followed by MS/MS scans also in the orbitrap(R=7500) on the 5 most abundant precursor ions with dynamic exclusion ofpreviously selected ions. Tandem mass spectra were interpreted bySEQUEST and additional manual control. The identified peptide sequencewas assured by comparison of the generated natural peptide fragmentationpattern with the fragmentation pattern of a synthetic sequence-identicalreference peptide.

Label-free relative LC-MS quantitation was performed by ion countingi.e. by extraction and analysis of LC-MS features (Mueller et al.,2007). The method assumes that the peptide's LC-MS signal areacorrelates with its abundance in the sample. Extracted features werefurther processed by charge state deconvolution and retention timealignment (Mueller et al., 2008; Sturm et al., 2008). Finally, all LC-MSfeatures were cross-referenced with the sequence identification resultsto combine quantitative data of different samples and tissues to peptidepresentation profiles. The quantitative data were normalized in atwo-tier fashion according to central tendency to account for variationwithin technical and biological replicates. Thus, each identifiedpeptide can be associated with quantitative data allowing relativequantification between samples and tissues. In addition, allquantitative data acquired for peptide candidates was inspected manuallyto assure data consistency and to verify the accuracy of the automatedanalysis. For each peptide, a presentation profile was calculatedshowing the mean sample presentation as well as replicate variations.The profiles juxtapose gallbladder cancer and cholangiocarcinoma samplesto a baseline of normal tissue samples. Presentation profiles ofexemplary over-presented peptides are shown in FIGS. 1A-1G. Presentationscores for exemplary peptides are shown in Table 8.

TABLE 8 Presentation scores. The table lists peptides thatare very highly over-presented on tumors comparedto a panel of normal tissues (+++), highly over-presented on tumors compared to a panel of normaltissues (++) or over-presented on tumors comparedto a panel of normal tissues (+). The panel ofnormal tissues considered relevant for comparisonwith tumors consisted of: adipose tissue, adrenalgland, artery, bone marrow, brain, central nerve,colon, duodenum, esophagus, eye, gallbladder,heart, kidney, liver, lung, lymph node,mononuclear white blood cells, pancreas,parathyroid gland, peripheral nerve, peritoneum,pituitary, pleura, rectum, salivary gland,skeletal muscle, skin, small intestine, spleen,stomach, thyroid gland, trachea, ureter, urinary bladder, vein. PeptideSEQ ID No. Sequence Presentation 1 YAAEIASAL +++ 2 AAYPEIVAV +++ 3EMDSTVITV +++ 4 FLLEAQNYL +++ 5 GLIDEVMVLL +++ 6 LLLPLLPPLSPS +++ 7LLLSDPDKVTI +++ 8 LSASLVRIL +++ 9 RLAKLTAAV +++ 10 SAFPFPVTVSL +++ 11SIIDFTVTM +++ 12 TILPGNLQSW +++ 13 VLPRAFTYV +++ 14 YGIEFVVGV +++ 15SVIDSLPEI +++ 17 VLYDNTQLQL +++ 19 TAYPQVVVV + 20 VLQDELPQL ++ 21IAFPTSISV +++ 24 ISAPLVKTL +++ 25 NLSETASTMAL +++ 26 TAQTLVRIL +++ 27ALAEQVQKA ++ 30 FASGLIHRV +++ 31 IAIPFLIKL ++ 32 YVISQVFEI +

Example 2

Expression Profiling of Genes Encoding the Peptides of the Invention

Over-presentation or specific presentation of a peptide on tumor cellscompared to normal cells is sufficient for its usefulness inimmunotherapy, and some peptides are tumor-specific despite their sourceprotein occurring also in normal tissues. Still, mRNA expressionprofiling adds an additional level of safety in selection of peptidetargets for immunotherapies. Especially for therapeutic options withhigh safety risks, such as affinity-matured TCRs, the ideal targetpeptide will be derived from a protein that is unique to the tumor andnot found on normal tissues.

RNA Sources and Preparation

Surgically removed tissue specimens were provided as indicated above(see Example 1) after written informed consent had been obtained fromeach patient. Tumor tissue specimens were snap-frozen immediately aftersurgery and later homogenized with mortar and pestle under liquidnitrogen. Total RNA was prepared from these samples using TRI Reagent(Ambion, Darmstadt, Germany) followed by a cleanup with RNeasy (QIAGEN,Hilden, Germany); both methods were performed according to themanufacturer's protocol.

Total RNA from healthy human tissues for RNASeq experiments was obtainedfrom: Asterand (Detroit, Mich., USA & Royston, Herts, UK), BioCat GmbH(Heidelberg, Germany), BioServe (Beltsville, Md., USA), CapitalBioScience Inc. (Rockville, Md., USA), Geneticist Inc. (Glendale,Calif., USA), Istituto Nazionale Tumori “Pascale” (Naples, Italy),ProteoGenex Inc. (Culver City, Calif., USA), University HospitalHeidelberg (Heidelberg, Germany).

Total RNA from tumor tissues for RNASeq experiments was obtained from:ProteoGenex Inc. (Culver City, Calif., USA), Tissue Solutions Ltd(Glasgow, UK), University Hospital Tübingen (Tübingen, Germany).

Quality and quantity of all RNA samples were assessed on an Agilent 2100Bioanalyzer (Agilent, Waldbronn, Germany) using the RNA 6000 PicoLabChip Kit (Agilent).

RNAseq Experiments

Gene expression analysis of—tumor and normal tissue RNA samples wasperformed by next generation sequencing (RNAseq) by CeGaT (Tübingen,Germany). Briefly, sequencing libraries are prepared using the IlluminaHiSeq v4 reagent kit according to the provider's protocol (IlluminaInc., San Diego, Calif., USA), which includes RNA fragmentation, cDNAconversion and addition of sequencing adaptors. Libraries derived frommultiple samples are mixed equimolar and sequenced on the Illumina HiSeq2500 sequencer according to the manufacturer's instructions, generating50 bp single end reads. Processed reads are mapped to the human genome(GRCh38) using the STAR software. Expression data are provided ontranscript level as RPKM (Reads Per Kilobase per Million mapped reads,generated by the software Cufflinks) and on exon level (total reads,generated by the software Bedtools), based on annotations of the ensemblsequence database (Ensembl77). Exon reads are normalized for exon lengthand alignment size to obtain RPKM values.

Exemplary expression profiles of source genes of the present inventionthat are highly over-expressed or exclusively expressed in gallbladdercancer and cholangiocarcinoma are shown in FIGS. 2A to A-2C. Expressionscores for further exemplary genes are shown in Table 1:

TABLE 1 Expression scores. The table lists peptides fromgenes that are very highly over-expressed intumors compared to a panel of normal tissues(+++), highly over-expressed in tumors comparedto a panel of normal tissues (++) or over-expressed in tumors compared to a panel of normaltissues (+). The baseline for this score wascalculated from measurements of the followingrelevant normal tissues: adipose tissue, adrenalgland, artery, bile duct, blood cells, bonemarrow, brain, cartilage, colon, esophagus, eye,gallbladder, salivary gland, heart, kidney, liver,lung, lymph node, pancreas, parathyroid gland,peripheral nerve, peritoneum, pituitary, pleura,rectum, skeletal muscle, skin, small intestine,spleen, stomach, thyroid gland, trachea, ureter,urinary bladder, and vein. In case expressiondata for several samples of the same tissue typewere available, the arithmetic mean of allrespective samples was used for the calculation. SEQ ID No SequenceGene Expression 5 GLIDEVMVLL +++ 8 LSASLVRIL +++ 13 VLPRAFTYV +++ 14YGIEFVVGV +++

Example 3

In Vitro Immunogenicity for MHC Class I Presented Peptides

In order to obtain information regarding the immunogenicity of theTUMAPs of the present invention, the inventors performed investigationsusing an in vitro T-cell priming assay based on repeated stimulations ofCD8+ T cells with artificial antigen presenting cells (aAPCs) loadedwith peptide/MHC complexes and anti-CD28 antibody. This way, theinventors could show immunogenicity for HLA-A*0201 restricted TUMAPs ofthe invention, demonstrating that these peptides are T-cell epitopesagainst which CD8+ precursor T cells exist in humans (Table 10).

In Vitro Priming of CD8+ T Cells

In order to perform in vitro stimulations by artificial antigenpresenting cells loaded with peptide-MHC complex (pMHC) and anti-CD28antibody, the inventors first isolated CD8+ T cells from fresh HLA-A*02leukapheresis products via positive selection using CD8 microbeads(Miltenyi Biotec, Bergisch-Gladbach, Germany) of healthy donors obtainedfrom the University clinics Mannheim, Germany, after informed consent.

PBMCs and isolated CD8+ lymphocytes were incubated in T-cell medium(TCM) until use consisting of RPMI-Glutamax (Invitrogen, Karlsruhe,Germany) supplemented with 10% heat inactivated human AB serum(PAN-Biotech, Aidenbach, Germany), 100 U/ml Penicillin/100 μg/mlStreptomycin (Cambrex, Cologne, Germany), 1 mM sodium pyruvate (CC Pro,Oberdorla, Germany), 20 μg/ml Gentamycin (Cambrex). 2.5 ng/ml IL-7(PromoCell, Heidelberg, Germany) and 10 U/ml IL-2 (Novartis Pharma,NOmberg, Germany) were also added to the TCM at this step.

Generation of pMHC/anti-CD28 coated beads, T-cell stimulations andreadout was performed in a highly defined in vitro system using fourdifferent pMHC molecules per stimulation condition and 8 different pMHCmolecules per readout condition.

The purified co-stimulatory mouse IgG2a anti human CD28 Ab 9.3 (Jung etal., 1987) was chemically biotinylated usingSulfo-N-hydroxysuccinimidobiotin as recommended by the manufacturer(Perbio, Bonn, Germany). Beads used were 5.6 μm diameter streptavidincoated polystyrene particles (Bangs Laboratories, Illinois, USA). pMHCused for positive and negative control stimulations were A*0201/MLA-001(peptide ELAGIGILTV (SEQ ID NO. 39) from modified Melan-A/MART-1) andA*0201/DDX5-001 (YLLPAIVHI from DDX5, SEQ ID NO. 40), respectively.

800.000 beads/200 μl were coated in 96-well plates in the presence of4×12.5 ng different biotin-pMHC, washed and 600 ng biotin anti-CD28 wereadded subsequently in a volume of 200 μl. Stimulations were initiated in96-well plates by co-incubating 1×10⁶ CD8+ T cells with 2×10⁵ washedcoated beads in 200 μl TCM supplemented with 5 ng/ml IL-12 (PromoCell)for 3 days at 37^(c)C. Half of the medium was then exchanged by freshTCM supplemented with 80 U/ml IL-2 and incubating was continued for 4days at 37° C. This stimulation cycle was performed for a total of threetimes. For the pMHC multimer readout using 8 different pMHC moleculesper condition, a two-dimensional combinatorial coding approach was usedas previously described (Andersen et al., 2012) with minor modificationsencompassing coupling to 5 different fluorochromes. Finally, multimericanalyses were performed by staining the cells with Live/dead near IR dye(Invitrogen, Karlsruhe, Germany), CD8-FITC antibody clone SK1 (BD,Heidelberg, Germany) and fluorescent pMHC multimers. For analysis, a BDLSRII SORP cytometer equipped with appropriate lasers and filters wasused. Peptide specific cells were calculated as percentage of total CD8+cells. Evaluation of multimeric analysis was done using the FlowJosoftware (Tree Star, Oregon, USA). In vitro priming of specificmultimer+CD8+ lymphocytes was detected by comparing to negative controlstimulations. Immunogenicity for a given antigen was detected if atleast one evaluable in vitro stimulated well of one healthy donor wasfound to contain a specific CD8+ T-cell line after in vitro stimulation(i.e. this well contained at least 1% of specific multimer+ among CD8+T-cells and the percentage of specific multimer+ cells was at least 10×the median of the negative control stimulations).

In Vitro Immunogenicity for Gallbladder Cancer and CholangiocarcinomaPeptides

For tested HLA class I peptides, in vitro immunogenicity could bedemonstrated by generation of peptide specific T-cell lines. Exemplaryflow cytometry results after TUMAP-specific multimer staining for 2peptides of the invention are shown in FIG. 3 together withcorresponding negative controls. Additional exemplary flow cytometryresults after TUMAP-specific multimer staining for three peptides of theinvention are shown in FIGS. 4A-4C together with corresponding negativecontrols. Results for five peptides from the invention are summarized inTable 10A. Additional results for four peptides of the invention aresummarized in Table 10B.

TABLE 10A in vitro immunogenicity of HLA class I peptidesof the invention Seq ID Sequence wells 33 ILGTEDLIVEV ++ 34 LLWGNLPEI ++35 GLIDEVMVL ++ 36 ILVDWLVQV ++ 38 KIQEILTQV ++ Exemplary results of invitro immunogenicity experiments performed by the applicant for thepeptides of the invention. <20% = +; 20%-49% = ++; 50%-69% = +++; ;>=70%= ++++

TABLE 10B In vitro immunogenicity of HLA class I peptides of theinvention Exemplary results of in vitro immunogenicity experimentsconducted by the applicant for HLA-A*02 restricted peptides of theinvention. SEQ ID No Sequence Wells positive [%] 6 LLLPLLPPLSPS + 13VLPRAFTYV ++++ 16 AVMTDLPVI + 25 NLSETASTMAL ++ Results of in vitroimmunogenicity experiments are indicated. Percentage of positive wellsand donors (among evaluable) are summarized as indicated <20% = +;20%-49% = ++; 50%-69% = +++; >=70% = ++++

Example 4

Synthesis of Peptides

All peptides were synthesized using standard and well-established solidphase peptide synthesis using the Fmoc-strategy. Identity and purity ofeach individual peptide have been determined by mass spectrometry andanalytical RP-HPLC. The peptides were obtained as white to off-whitelyophilizes (trifluoro acetate salt) in purities of >50%. All TUMAPs arepreferably administered as trifluoro-acetate salts or acetate salts,other salt-forms are also possible.

Example 5

MHC Binding Assays

Candidate peptides for T cell based therapies according to the presentinvention were further tested for their MHC binding capacity (affinity).The individual peptide-MHC complexes were produced by UV-ligandexchange, where a UV-sensitive peptide is cleaved upon UV-irradiation,and exchanged with the peptide of interest as analyzed.

Only peptide candidates that can effectively bind and stabilize thepeptide-receptive MHC molecules prevent dissociation of the MHCcomplexes. To determine the yield of the exchange reaction, an ELISA wasperformed based on the detection of the light chain (β2m) of stabilizedMHC complexes. The assay was performed as generally described in Rodenkoet al. (Rodenko et al., 2006).

96 well MAXISorp plates (NUNC) were coated over night with 2 ug/mlstreptavidin in PBS at room temperature, washed 4× and blocked for 1h at37° C. in 2% BSA containing blocking buffer. RefoldedHLA-A*02:01/MLA-001 monomers served as standards, covering the range of15-500 ng/ml. Peptide-MHC monomers of the UV-exchange reaction werediluted 100-fold in blocking buffer. Samples were incubated for 1 h at37° C., washed four times, incubated with 2 ug/ml HRP conjugatedanti-β2m for 1h at 37° C., washed again and detected with TMB solutionthat is stopped with NH₂SO₄. Absorption was measured at 450 nm.Candidate peptides that show a high exchange yield (preferably higherthan 50%, most preferred higher than 75%) are generally preferred for ageneration and production of antibodies or fragments thereof, and/or Tcell receptors or fragments thereof, as they show sufficient avidity tothe MHC molecules and prevent dissociation of the MHC complexes.

TABLE 11 MHC class I binding scores. SEQ ID No Sequence Peptide exchange1 YAAEIASAL +++ 2 AAYPEIVAV +++ 3 EMDSTVITV +++ 4 FLLEAQNYL ++++ 5GLIDEVMVLL ++++ 6 LLLPLLPPLSPS ++++ 7 LLLSDPDKVTI ++++ 8 LSASLVRIL + 9RLAKLTAAV ++++ 10 SAFPFPVTVSL ++ 11 SIIDFTVTM ++++ 12 TILPGNLQSW + 13VLPRAFTYV ++++ 14 YGIEFVVGV ++++ 15 SVIDSLPEI ++++ 16 AVMTDLPVI ++++ 17VLYDNTQLQL ++++ 18 SLSPDLSQV +++ 19 TAYPQVVVV ++ 20 VLQDELPQL ++++ 21IAFPTSISV +++ 22 SAFGFPVIL ++ 23 SLLSELLGV ++++ 24 ISAPLVKTL + 25NLSETASTMAL ++++ 26 TAQTLVRIL + 27 ALAEQVQKA +++ 28 YASGSSASL + 29FASEVSNVL ++++ 30 FASGLIHRV +++ 31 IAIPFLIKL ++++ 32 YVISQVFEI ++++Binding of HLA-class I restricted peptides to HLA-A*02:01 was ranged bypeptide exchange yield: >10% = +; >20% = ++; >50 = +++; >75% = ++++

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1. A method for killing target cells in a patient who has gallbladdercancer and cholangiocarcinoma, comprising administering to the patient apopulation of activated T cells that kill a target cell that presents apeptide consisting of the amino acid sequence of SEQ ID NO: 3 on thecell surface.
 2. The method of claim 1, wherein the T cells areautologous to the patient.
 3. The method of claim 1, wherein the T cellsare obtained from a healthy donor.
 4. The method of claim 1, wherein theactivated T cells are produced by contacting T cells with the peptideloaded human class I or II MHC molecules expressed on the surface of anantigen-presenting cell for a period of time sufficient to activate theT cells.
 5. The method of claim 1, wherein the activated T cells areexpanded in vitro.
 6. The method of claim 1, wherein the peptide is in acomplex with an MHC class I molecule.
 7. The method of claim 4, whereinthe antigen presenting cell is infected with a recombinant virusexpressing the peptide.
 8. The method of claim 7, wherein the antigenpresenting cell is a dendritic cell or a macrophage.
 9. The method ofclaim 5, wherein the expansion is in the presence of an anti-CD28antibody and IL-12.
 10. The method of claim 1, wherein the population ofactivated T cells comprises CD8-positive cells.
 11. The method of claim4, wherein the contacting is in vitro.
 12. The method of claim 1,wherein the population of activated T cells is administered in the formof a composition.
 13. The method of claim 12, wherein the compositioncomprises an adjuvant.
 14. The method of claim 13, wherein the adjuvantis selected from anti-CD40 antibody, imiquimod, resiquimod, GM-CSF,cyclophosphamide, sunitinib, bevacizumab, interferon-alpha,interferon-beta, CpG oligonucleotides and derivatives, poly-(I:C) andderivatives, RNA, sildenafil, particulate formulations with poly(lactideco-glycolide) (PLG), virosomes, interleukin (IL)-1, IL-2, IL-4, IL-7,IL-12, IL-13, IL-15, IL-21, and IL-23.
 15. The method of claim 14,wherein the adjuvant comprises IL-2.
 16. The method of claim 14, whereinthe adjuvant comprises IL-7.
 17. The method of claim 14, wherein theadjuvant comprises IL-15.
 18. The method of claim 14, wherein theadjuvant comprises IL-21.
 19. A method of treating a patient who hasgallbladder cancer and cholangiocarcinoma, comprising administering tothe patient a composition comprising a population of activated T cells,wherein the activated T cells kill cancer cells in the patient, whereinthe cancer cells present a peptide consisting of the amino acid sequenceof SEQ ID NO:
 3. 20. The method of claim 19, wherein the T cells areautologous to the patient.